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10. A method of modulating the level of WRKY protein in a plant,
comprising: a) introducing into a plant cell a recombinant expression
cassette comprising a WRKY polynucleotide of claim 1 operably linked to a
promoter; b) culturing the plant cell under plant growing conditions to
produce a regenerated plant; and c) inducing expression of said
polynucleotide for a time sufficient to modulate the WRKY protein in said
plant.

20. A method of modulating the level of WRKY protein in a plant,
comprising: a) introducing into a plant cell a recombinant expression
cassette comprising the polynucleotide of claim 12 operably linked to a
promoter; b) culturing the plant cell under plant growing conditions to
produce a regenerated plant; and c) inducing expression of said
polynucleotide for a time sufficient to modulate WRKY protein in said
plant.

29. A method of modulating the level of WRKY protein in a plant,
comprising: a) introducing into a plant cell a recombinant expression
cassette comprising the polynucleotide of claim 21 operably linked to a
promoter; b) culturing the plant cell under plant growing conditions to
produce a regenerated plant; and c) inducing expression of said
polynucleotide for a time sufficient to modulate WRKY protein in said
plant.

31. A method of regulating transcription of a heterologous nucleic acid
comprising the steps of: a) introducing into a plant cell the
polynucleotide of claim 30 operably linked to a heterologous nucleic
acid; b) culturing the plant cell under plant growing conditions to
produce a regenerated plant; and c) inducing expression of the
heterologous nucleic acid.

36. An isolated transcriptional region that is capable of driving
transcription in a plant, wherein the transcriptional region comprises
the polynucleotide shown in SEQ ID NO: 35.

37. A method of regulating the SA-dependent SAR response in a plant
comprising the steps of: a) introducing into a plant cell a recombinant
expression cassette comprising the polynucleotide of claim 1 operably
linked to a promoter; b) culturing the plant cell under plant growing
conditions to produce a regenerated plant; and c) inducing expression of
said polynucleotide for a time sufficient to modulate the SA-dependent
SAR response.

38. The method of claim 37, wherein the polynucleotide is shown in SEQ ID
NO: 1.

39. The method of claim 38, wherein the polynucleotide is in the antisense
orientation.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. provisional application
No. 60/190,950, filed Mar. 21, 2000, which is herein incorporated by
reference.

BACKGROUND OF THE INVENTION

[0002] Plant disease outbreaks have resulted in catastrophic crop failures
that have triggered famines and caused major social change. Generally,
the best strategy for plant disease control is to use resistant cultivars
selected or developed by plant breeders for this purpose. However, the
potential for serious crop disease epidemics persists today, as evidenced
by outbreaks of the Victoria blight of oats and southern corn leaf
blight. Accordingly, molecular methods are needed to supplement
traditional breeding methods to protect plants from pathogen attack.

[0003] A host of cellular processes enables plants to defend themselves
from disease caused by pathogenic agents. These processes apparently form
an integrated set of resistance mechanisms that is activated by initial
infection and then limits further spread of the invading pathogenic
microorganism.

[0004] WRKY proteins are a family of plant-specific zinc-finger-type
factors implicated in the regulation of genes associated with a plant's
response to a pathogen or stress, such as wounding. In addition, WRKY
proteins have been implicated in senescence, trichome development and the
biosynthesis of secondary metabolites. In parsley, WRKY proteins have
been found to bind specifically to functionally defined TGAC-containing W
box promoter elements within the Pathogenesis-Related Class 10 (PR-10)
genes. The WRKY proteins in parsley are rapidly and locally activated in
leaf tissue around the infection site of a pathogen. Transient expression
studies in parsley protoplasts showed that a specific arrangement of W
box elements in the WRKY1 promoter itself is necessary and sufficient for
early activation and that WRKY1 binds to such elements (Rushton, et al.,
EMBO Journal, 15(2):5690-5700 (1996)).

[0005] WRKY proteins have been classified into three groups. Group I
typically has two WRKY domains of a unique zinc-finger-like motif. Group
II typically has only one WRKY domain. Group III has one WRKY domain but
instead of the C.sub.2-H.sub.2 motif found in Groups I and II, the WRKY
domain in Group III has a C.sub.2-HC motif.

[0006] The present invention discloses WRKY polynucleotides from
sunflower, maize, rice, wheat and soybean. WRKY polynucleotides may be
used to engineer plants to resist pathogens and to survive stress. In
addition, WRKY cDNA clones and DNA segments of genomic DNA, and their
homologs and derivatives, may be used as molecular probes to track
inheritance of corresponding loci in genetic crosses, and thus facilitate
the plant breeding process. Moreover, these DNA sequences may also be
used as probes to isolate, identify and genetically map WRKY and other
closely related disease resistance genes. Further the polynucleotides of
the present invention, either as a full-length or a sub-sequence, could
be used to find genes and their promoters that respond to a WRKY domain.

[0007] The present invention also discloses a transcriptional regulatory
region sequence from a sunflower WRKY gene, which can induce expression
of a gene of interest during pathogen infection or in the presence of
oxalic acid or salicylic acid. Gene expression encompasses a number of
steps from DNA template to the final protein or protein product.
Initiation of transcription of a gene is generally understood to be the
predominant controlling factor in determining expression of a gene.

[0008] Controlling the expression of agronomic genes in transgenic plants
is considered by those skilled in the art to provide several advantages
over generalized or constitutive expression. The ability to control gene
expression may be utilized to time expression for when a pathogen attacks
a plant thus avoiding certain regulatory and commercial issues. A
pathogen or chemically-inducible promoter can reduce potential yield loss
by limiting expression of some pernicious, yet useful agronomic genes to
only when it is needed. Further advantages of utilizing promoters that
function in an inducible manner include reduced resource drain on the
plant in making a gene product constitutively. Said gene products may
include general toxin degradative genes such as oxalate oxidase or other
disease resistance genes. There is a need in the art for novel promoters
capable of driving pathogen or chemical-inducible gene expression in
plants. It is considered important by those skilled in the art to
continue to provide pathogen or chemical-inducible transcriptional
regulatory regions capable of driving expression of genes that may confer
a selective advantage to a plant.

SUMMARY OF THE INVENTION

[0009] Generally, it is the object of the present invention to provide
nucleic acids and proteins relating to WRKY. It is an object of the
present invention to provide transgenic plants comprising the nucleic
acids of the present invention. It is another object of the present
invention to provide methods for modulating, in a transgenic plant, the
expression of the nucleic acids of the present invention.

[0010] Therefore, in one aspect, the present invention relates to an
isolated nucleic acid comprising a member selected from the group
consisting of (a) a polynucleotide encoding a polypeptide of the present
invention; (b) a polynucleotide having at least 75 or 80% sequence
identity to the polynucleotides of the present invention; (c) a
polynucleotide that hybridizes under high stringency conditions to the
polynucleotides of the present invention; and (d) a polynucleotide
complementary to a polynucleotide of (a) through (c). The isolated
nucleic acid can be DNA. The isolated nucleic acid can also be RNA.

[0011] In another aspect, the present invention relates to vectors
comprising the polynucleotides of the present invention. Also the present
invention relates to recombinant expression cassettes, comprising a
nucleic acid of the present invention operably linked to a promoter.

[0012] In another aspect, the present invention is directed to a host cell
into which has been introduced the recombinant expression cassette.

[0013] In yet another aspect, the present invention relates to a
transgenic plant or plant cell comprising a recombinant expression
cassette with a promoter operably linked to any of the isolated nucleic
acids of the present invention. Preferred plants containing the
recombinant expression cassette of the present invention include but are
not limited to maize, soybean, sunflower, sorghum, canola, wheat,
alfalfa, cotton, rice barley, and millet. The present invention also
provides transgenic seed from the transgenic plant.

[0014] In another aspect, the present invention relates to an isolated
protein selected from the group consisting of (a) a polypeptide
comprising at least 40 or 50 contiguous amino acids of a polypeptide of
the present invention; (b) a polypeptide comprising at least 75 or 80%
sequence identity to a polypeptide of the present invention; (c) a
polypeptide encoded by a nucleic acid of the present invention; and (d) a
polypeptide characterized by a polypeptide of the present invention.

[0015] In a further aspect, the present invention relates to a method of
modulating the level of protein in a plant by introducing into a plant
cell a recombinant expression cassette comprising a polynucleotide of the
present invention operably linked to a promoter; culturing the plant cell
under plant growing conditions to produce a regenerated plant; and
inducing expression of the polynucleotide for a time sufficient to
modulate the protein of the present invention in the plant. Preferred
plants of the present invention include but are not limited to maize,
soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice,
barley, and millet. The level of protein in the plant can either be
increased or decreased.

[0016] In addition, the present invention provides a transcriptional
regulatory region capable of directing pathogen or chemical-induced gene
expression. Further, the present invention provides for plants, plant
cells, and seeds from the plant containing the transcriptional regulatory
region. The present invention also provides for a method of expressing a
heterologous nucleic acid during pathogen infection or upon chemical
induction with the transcriptional regulatory region of the present
invention.

[0057] The present invention provides, among other things, compositions
and methods for modulating (i.e., increasing or decreasing) the level of
polynucleotides and polypeptides of the present invention in plants. In
particular, the polynucleotides and polypeptides of the present invention
can be expressed temporally or spatially, e.g., at developmental stages,
in tissues, and/or in quantities, which are uncharacteristic of
non-recombinantly engineered plants. The transcriptional regulatory
region of a WRKY polynucleotide, such as the sunflower WRKY1-2
polynucleotide (SEQ ID NO: 35), can be used to drive expression of a gene
of interest during pathogen infection or by chemical induction. Thus, the
present invention provides utility in such exemplary applications as
disease resistance.

[0058] The present invention also provides isolated nucleic acid
comprising polynucleotides of sufficient length and complementarity to a
gene of the present invention to use as probes or amplification primers
in the detection, quantitation, or isolation of gene transcripts. For
example, isolated nucleic acids of the present invention can be used as
probes in detecting deficiencies in the level of mRNA in screenings for
desired transgenic plants, for detecting mutations in the gene (e.g.,
substitutions, deletions, or additions), for monitoring upregulation of
expression or changes in enzyme activity in screening assays of
compounds, for detection of any number of allelic variants
(polymorphisms), orthologs, or paralogs of the gene, or for site directed
mutagenesis in eukaryotic cells (see, e.g., U.S. Pat. No. 5,565,350). The
isolated nucleic acids of the present invention can also be used for
recombinant expression of their encoded polypeptides, or for use as
immunogens in the preparation and/or screening of antibodies. The
isolated nucleic acids of the present invention can also be employed for
use in sense or antisense suppression of one or more genes of the present
invention in a host cell, tissue, or plant. Attachment of chemical
agents, which bind, intercalate, cleave and/or crosslink to the isolated
nucleic acids of the present invention can also be used to modulate
transcription or translation. In addition, the present invention relates
to finding genes and promoters that respond to WRKY domains. The
full-length sequence of WRKY or a subsequence of WRKY could be used alone
or fused to additional sequence to determine genes and promoter that
respond to WRKY domains. The present invention also provides isolated
proteins comprising a polypeptide of the present invention (e.g.,
preproenzyme, proenzyme, or enzymes).

[0061] Assays that measure antipathogenic activity are commonly known in
the art, as are methods to quantify disease resistance in plants
following pathogen infection. See, for example, U.S. Pat. No. 5,614,395,
herein incorporated by reference. Such techniques include, measuring over
time, the average lesion diameter, the pathogen biomass, and the overall
percentage of decayed plant tissues. For example, a plant either
expressing an antipathogenic polypeptide or having an antipathogenic
composition applied to its surface shows a decrease in tissue necrosis
(i.e., lesion diameter) or a decrease in plant death following pathogen
challenge when compared to a control plant that was not exposed to the
antipathogenic composition. Alternatively, antipathogenic activity can be
measured by a decrease in pathogen biomass. For example, a plant
expressing an antipathogenic polypeptide or exposed to an antipathogenic
composition is challenged with a pathogen of interest. Over time, tissue
samples from the pathogen-inoculated tissues are obtained and RNA is
extracted. The percent of a specific pathogen RNA transcript relative to
the level of a plant specific transcript allows the level of pathogen
biomass to be determined. See, for example, Thomma et al. (1998) Plant
Biology 95:15107-15111, herein incorporated by reference.

[0062] Furthermore, in vitro antipathogenic assays include, for example,
the addition of varying concentrations of the antipathogenic composition
to paper disks and placing the disks on agar containing a suspension of
the pathogen of interest. Following incubation, clear inhibition zones
develop around the discs that contain an effective concentration of the
antipathogenic polypeptide (Liu et al. (1994) Plant Biology 91:1888-1892,
herein incorporated by reference). Additionally, microspectrophotometrica-
l analysis can be used to measure the in vitro antipathogenic properties
of a composition (Hu et al. (1997) Plant Mol. Biol. 34:949-959 and Cammue
et al. (1992) J. Biol. Chem. 267: 2228-2233, both of which are herein
incorporated by reference).

[0063] Plasmids containing the polynucleotide sequences of the invention
were deposited with American Type Culture Collection (ATCC), Manassas,
Va., and assigned the following Patent Deposit Designation numbers: for
maize ZmWRKY3-1 the designation is PTA-1590; for SWRKY1-1 the designation
is PTA-1510, for SWRKY1-2 the designation is PTA-1504, for SWRKY1-3 the
designation is PTA-1511, for SWRKY1-4 the designation is PTA-1509, and
for the 5' regulatory region of WRKY1-2 the designation is PTA-1505.
These deposits will be maintained under the terms of the Budapest Treaty
on the International Recognition of the Deposit of Microorganisms for the
Purposes of Patent Procedure. These deposits were made merely as a
convenience for those of skill in the art and are not an admission that a
deposit is required under 35 U.S.C. .sctn. 112.

[0064] Definitions

[0065] Units, prefixes, and symbols may be denoted in their SI accepted
form. Unless otherwise indicated, nucleic acids are written left to right
in 5' to 3' orientation, amino acid sequences are written left to right
in amino to carboxy orientation, respectively. Numeric ranges are
inclusive of the numbers defining the range and include each integer
within the defined range. Amino acids may be referred to herein by either
their commonly known three letter symbols or by the one-letter symbols
recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
Nucleotides, likewise, may be referred to by their commonly accepted
single-letter codes. The terms defined below are more fully defined by
reference to the specification as a whole.

[0067] As used herein, "antisense orientation" includes reference to a
duplex polynucleotide sequence, which is operably linked to a promoter in
an orientation where the antisense strand is transcribed. The antisense
strand is sufficiently complementary to an endogenous transcription
product such that translation of the endogenous transcription product is
often inhibited.

[0068] By "encoding" or "encoded", with respect to a specified nucleic
acid, is meant comprising the information for translation into the
specified protein. A nucleic acid encoding a protein may comprise
non-translated sequences (e.g., introns) within translated regions of the
nucleic acid, or may lack such intervening non-translated sequences
(e.g., as in cDNA). The information by which a protein is encoded is
specified by the use of codons. Typically, the amino acid sequence is
encoded by the nucleic acid using the "universal" genetic code. However,
variants of the universal code, such as are present in some plant,
animal, and fingal mitochondria, the bacterium Mycoplasma capricolum, or
the ciliate Macronucleus, may be used when the nucleic acid is expressed
therein.

[0069] When the nucleic acid is prepared or altered synthetically,
advantage can be taken of known codon preferences of the intended host
where the nucleic acid is to be expressed. For example, although nucleic
acid sequences of the present invention may be expressed in both
monocotyledonous and dicotyledonous plant species, sequences can be
modified to account for the specific codon preferences and GC content
preferences of monocotyledons or dicotyledons as these preferences have
been shown to differ (Murray et al. Nucl. Acids Res. 17:477-498 (1989)).
Thus, the maize preferred codon for a particular amino acid might be
derived from known gene sequences from maize. Maize codon usage for 28
genes from maize plants is listed in Table 4 of Murray et al., supra.

[0070] As used herein, "heterologous" in reference to a nucleic acid is a
nucleic acid that originates from a foreign species, or, if from the same
species, is substantially modified from its native form in composition
and/or genomic locus by deliberate human intervention. For example, a
promoter operably linked to a heterologous structural gene is from a
species different from that from which the structural gene was derived,
or, if from the same species, one or both are substantially modified from
their original form. A heterologous protein may originate from a foreign
species, or, if from the same species, is substantially modified from its
original form by deliberate human intervention.

[0071] By "host cell" is meant a cell, which contains a vector and
supports the replication and/or expression of the vector. Host cells may
be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast,
insect, amphibian, or mammalian cells. Preferably, host cells are
monocotyledonous or dicotyledonous plant cells. A particularly preferred
monocotyledonous host cell is a maize host cell.

[0072] The term "introduced" in the context of inserting a nucleic acid
into a cell, means "transfection" or "transformation" or "transduction"
and includes reference to the incorporation of a nucleic acid into a
eukaryotic or prokaryotic cell where the nucleic acid may be incorporated
into the genome of the cell (e.g., chromosome, plasmid, plastid or
mitochondrial DNA), converted into an autonomous replicon, or transiently
expressed (e.g., transfected mRNA).

[0073] The terms "isolated" refers to material, such as a nucleic acid or
a protein, which is: (1) substantially or essentially free from
components that normally accompany or interact with it as found in its
naturally occurring environment. The isolated material optionally
comprises material not found with the material in its natural
environment; or (2) if the material is in its natural environment, the
material has been synthetically (non-naturally) altered by deliberate
human intervention to a composition and/or placed at a location in the
cell (e.g., genome or subcellular organelle) not native to a material
found in that environment. The alteration to yield the synthetic material
can be performed on the material within or removed from its natural
state. For example, a naturally occurring nucleic acid becomes an
isolated nucleic acid if it is altered, or if it is transcribed from DNA
which has been altered, by means of human intervention performed within
the cell from which it originates. See, e.g., Compounds and Methods for
Site Directed Mutagenesis in Eukaryotic Cells, Kmiec, U.S. Pat. No.
5,565,350; In Vivo Homologous Sequence Targeting in Eukaryotic Cells;
Zarling et al., PCT/US93/03868. Likewise, a naturally occurring nucleic
acid (e.g., a promoter) becomes isolated if it is introduced by
non-naturally occurring means to a locus of the genome not native to that
nucleic acid. Nucleic acids, which are "isolated", as defined herein, are
also referred to as "heterologous" nucleic acids.

[0074] As used herein, "nucleic acid" includes reference to a
deoxyribonucleotide or ribonucleotide polymer in either single- or
double-stranded form, and unless otherwise limited, encompasses known
analogues having the essential nature of natural nucleotides in that they
hybridize to single-stranded nucleic acids in a manner similar to
naturally occurring nucleotides (e.g., peptide nucleic acids).

[0076] As used herein "operably linked" includes reference to a functional
linkage between a promoter and a second sequence, wherein the promoter
sequence initiates and mediates transcription of the DNA sequence
corresponding to the second sequence. Generally, operably linked means
that the nucleic acid sequences being linked are contiguous and, where
necessary to join two protein coding regions, contiguous and in the same
reading frame.

[0077] As used herein, the term "plant" includes reference to whole
plants, plant organs (e.g., leaves, stems, roots, etc.), seeds and plant
cells and progeny of same. Plant cell, as used herein includes, without
limitation, seeds, suspension cultures, embryos, meristematic regions,
callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen,
and microspores. The class of plants, which can be used in the methods of
the invention, is generally as broad as the class of higher plants
amenable to transformation techniques, including both monocotyledonous
and dicotyledonous plants. Preferred plants include, but are not limited
to maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton,
rice, barley, and millet. A particularly preferred plant is maize (Zea
mays).

[0078] As used herein, "polynucleotide" includes reference to a
deoxyribopolynucleotide, ribopolynucleotide, or analogs thereof that have
the essential nature of a natural ribonucleotide in that they hybridize,
under stringent hybridization conditions, to substantially the same
nucleotide sequence as naturally occurring nucleotides and/or allow
translation into the same amino acid(s) as the naturally occurring
nucleotide(s). A polynucleotide can be full-length or a subsequence of a
native or heterologous structural or regulatory gene. Unless otherwise
indicated, the term includes reference to the specified sequence as well
as the complementary sequence thereof. Thus, DNAs or RNAs with backbones
modified for stability or for other reasons are "polynucleotides" as that
term is intended herein. Moreover, DNAs or RNAs comprising unusual bases,
such as inosine, or modified bases, such as tritylated bases, to name
just two examples, are polynucleotides as the term is used herein. It
will be appreciated that a great variety of modification have been made
to DNA and RNA that serve many useful purposes known to those of skill in
the art. The term polynucleotide as it is employed herein embraces such
chemically, enzymatically or metabolically modified forms of
polynucleotides, as well as the chemical forms of DNA and RNA
characteristic of viruses and cells, including among other things, simple
and complex cells.

[0079] The terms "polypeptide", "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid residues. The
terms apply to amino acid polymers in which one or more amino acid
residue is an artificial chemical analogue of a corresponding naturally
occurring amino acid, as well as to naturally occurring amino acid
polymers. The essential nature of such analogues of naturally occurring
amino acids is that, when incorporated into a protein, that protein is
specifically reactive to antibodies elicited to the same protein but
consisting entirely of naturally occurring amino acids. The terms
"polypeptide", "peptide", and "protein" are also inclusive of
modifications including, but not limited to, glycosylation, lipid
attachment, sulfation, gamma-carboxylation of glutamic acid residues,
hydroxylation and ADP-ribosylation. It will be appreciated, as is well
known and as noted above, that polypeptides are not always entirely
linear. For instance, polypeptides may be branched as a result of
ubiquination, and they may be circular, with or without branching,
generally as a result of post-translation events, including natural
processing event and events brought about by human manipulation which do
not occur naturally. Circular, branched and branched circular
polypeptides may be synthesized by non-translation natural process and by
entirely synthetic methods, as well. Further, this invention contemplates
the use of both the methionine containing and the methionine-less amino
terminal variants of the protein of the invention.

[0080] As used herein "promoter or transcriptional regulatory region"
includes reference to a region of DNA upstream from the start of
transcription and involved in recognition and binding of RNA polymerase
and other proteins to initiate transcription. A "plant promoter or
transcriptional regulatory region" is a promoter or transcriptional
regulatory region capable of initiating transcription in plant cells
whether or not its origin is a plant cell. Exemplary plant promoters
include, but are not limited to, those that are obtained from plants,
plant viruses, and bacteria which comprise genes expressed in plant cells
such as Agrobacterium or Rhizobium. Examples of promoters under
developmental control include promoters that preferentially initiate
transcription in certain tissues, such as leaves, roots, or seeds. Such
promoters are referred to as "tissue preferred". Promoters who initiate
transcription only in certain tissue are referred to as "tissue
specific". A "cell type" specific promoter primarily drives expression in
certain cell types in one or more organs, for example, vascular cells in
roots or leaves. An "inducible" or "repressible" promoter is a promoter,
which is under environmental control. Examples of environmental
conditions that may effect transcription by inducible promoters include
anaerobic conditions or the presence of light. Tissue specific, tissue
preferred, cell type specific, and inducible promoters constitute the
class of "non-constitutive" promoters. A "constitutive" promoter is a
promoter, which is active under most environmental conditions.

[0081] As used herein "recombinant" includes reference to a cell or
vector, that has been modified by the introduction of a heterologous
nucleic acid or that the cell is derived from a cell so modified. Thus,
for example, recombinant cells express genes that are not found in
identical form within the native (non-recombinant) form of the cell or
express native genes that are otherwise abnormally expressed,
under-expressed or not expressed at all as a result of deliberate human
intervention. The term "recombinant" as used herein does not encompass
the alteration of the cell or vector by naturally occurring events (e.g.,
spontaneous mutation, natural transformation/transduction/transposition)
such as those occurring without deliberate human intervention.

[0082] As used herein, a "recombinant expression cassette" is a nucleic
acid construct, generated recombinantly or synthetically, with a series
of specified nucleic acid elements, which permit transcription of a
particular nucleic acid in a host cell. The recombinant expression
cassette can be incorporated into a plasmid, chromosome, mitochondrial
DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the
recombinant expression cassette portion of an expression vector includes,
among other sequences, a nucleic acid to be transcribed, and a promoter.

[0083] The term "residue" or "amino acid residue" or "amino acid" are used
interchangeably herein to refer to an amino acid that is incorporated
into a protein, polypeptide, or peptide (collectively "protein"). The
amino acid may be a naturally occurring amino acid and, unless otherwise
limited, may encompass non-natural analogs of natural amino acids that
can function in a similar manner as naturally occurring amino acids.

[0084] The term "selectively hybridizes" includes a reference to
hybridization, under stringent hybridization conditions, of a nucleic
acid sequence to a specified nucleic acid target sequence to a detectably
greater degree (e.g., at least 2-fold over background) than its
hybridization to non-target nucleic acid sequences and to the substantial
exclusion of non-target nucleic acids. Selectively hybridizing sequences
typically have about at least 80% sequence identity, preferably 90%
sequence identity, and most preferably 100% sequence identity (i.e.,
complementary) with each other.

[0085] The terms "stringent conditions" or "stringent hybridization
conditions" include reference to conditions under which a probe will
hybridize to its target sequence, to a detectably greater degree than
other sequences (e.g., at least 2-fold over background). Stringent
conditions are sequence-dependent and will be different in different
circumstances. By controlling the stringency of the hybridization and/or
washing conditions, target sequences can be identified which are 100%
complementary to the probe (homologous probing). Alternatively,
stringency conditions can be adjusted to allow some mismatching in
sequences so that lower degrees of similarity are detected (heterologous
probing). Generally, a probe is less than about 1000 nucleotides in
length, optionally less than 500 nucleotides in length.

[0086] Typically, stringent conditions will be those in which the salt
concentration is less than about 1.5 M Na ion, typically about 0.01 to
1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the
temperature is at least about 30.degree. C. for short probes (e.g., 10 to
50 nucleotides) and at least about 60.degree. C. for long probes (e.g.,
greater than 50 nucleotides). Stringent conditions may also be achieved
with the addition of destabilizing agents such as formamide. Exemplary
low stringency conditions include hybridization with a buffer solution of
30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at
37.degree. C., and a wash in 1.times. to 2.times. SSC (20.times. SSC =3.0
M NaCl/0.3 M trisodium citrate) at 50 to 55.degree. C. Exemplary moderate
stringency conditions include hybridization in 40 to 45% formamide, 1 M
NaCl, 1% SDS at 37.degree. C., and a wash in 0.5.times. to 1.times. SSC
at 55 to 60.degree. C. Exemplary high stringency conditions include
hybridization in 50% formamide, I M NaCl, 1% SDS at 37.degree. C., and a
wash in0.1.times. SSC at 60 to 65.degree. C.

[0087] Specificity is typically the function of post-hybridization washes,
the critical factors being the ionic strength and temperature of the
final wash solution. For DNA-DNA hybrids, the T.sub.m can be approximated
from the equation of Meinkoth and Wahl, Anal. Biochem., 138:267-284
(1984): T.sub.m=81.5.degree. C.+16.6 (log M)+0.41 (%CG)-0.61 (%
form)-500/L; where M is the molarity of monovalent cations, %CG is the
percentage of guanosine and cytosine nucleotides in the DNA, % form is
the percentage of formamide in the hybridization solution, and L is the
length of the hybrid in base pairs. The T.sub.m is the temperature (under
defined ionic strength and pH) at which 50% of a complementary target
sequence hybridizes to a perfectly matched probe. T.sub.m is reduced by
about 1.degree. C. for each 1% of mismatching; thus, T.sub.m,
hybridization and/or wash conditions can be adjusted to hybridize to
sequences of the desired identity. For example, if sequences with
.gtoreq.90% identity are sought, the T.sub.m can be decreased 10.degree.
C. Generally, stringent conditions are selected to be about 5.degree. C.
lower than the thermal melting point (T.sub.m) for the specific sequence
and its complement at a defined ionic strength and pH. However, severely
stringent conditions can utilize a hybridization and/or wash at 1, 2, 3,
or 4.degree. lower than the thermal melting point (T.sub.m); moderately
stringent conditions can utilize a hybridization and/or wash at 6, 7, 8,
9, or 10.degree. C. lower than the thermal melting point (T.sub.m); low
stringency conditions can utilize a hybridization and/or wash at 11, 12,
13, 14, 15, or 20.degree. C. lower than the thermal melting point
(T.sub.m). Using the equation, hybridization and wash compositions, and
desired T.sub.m, those of ordinary skill will understand that variations
in the stringency of hybridization and/or wash solutions are inherently
described. If the desired degree of mismatching results in a T.sub.m of
less than 45.degree. C. (aqueous solution) or 32.degree. C. (formamide
solution) it is preferred to increase the SSC concentration so that a
higher temperature can be used. An extensive guide to the hybridization
of nucleic acids is found in Tijssen, Laboratory Techniques in
Biochemistry and Molecular Biology--Hybridization with Nucleic Acid
Probes, Part I, Chapter 2 "Overview of principles of hybridization and
the strategy of nucleic acid probe assays", Elsevier, New York (1993);
and Current Protocols in Molecular Biology, Chapter 2, Ausubel, et al.,
Eds., Greene Publishing and Wiley-Interscience, New York (1995).

[0088] As used herein, "transgenic plant" includes reference to a plant,
which comprises within its genome a heterologous polynucleotide.
Generally, the heterologous polynucleotide is stably integrated within
the genome such that the polynucleotide is passed on to successive
generations. The heterologous polynucleotide may be integrated into the
genome alone or as part of a recombinant expression cassette.
"Transgenic" is used herein to include any cell, cell line, callus,
tissue, plant part or plant, the genotype of which has been altered by
the presence of heterologous nucleic acid including those transgenics
initially so altered as well as those created by sexual crosses or
asexual propagation from the initial transgenic. The term "transgenic" as
used herein does not encompass the alteration of the genome (chromosomal
or extra-chromosomal) by conventional plant breeding methods or by
naturally occurring events such as random cross-fertilization,
non-recombinant viral infection, non-recombinant bacterial
transformation, non-recombinant transposition, or spontaneous mutation.

[0089] As used herein, "vector" includes reference to a nucleic acid used
in transfection of a host cell and into which can be inserted a
polynucleotide. Vectors are often replicons. Expression vectors permit
transcription of a nucleic acid inserted therein.

[0090] The following terms are used to describe the sequence relationships
between two or more nucleic acids or polynucleotides: (a) "reference
sequence", (b) "comparison windows", (c) "sequence identity", (d)
"percentage of sequence identity", and (e) "substantial identity".

[0091] (a) As used herein, "reference sequence" is a defined sequence used
as a basis for sequence comparison. A reference sequence may be a subset
or the entirety of a specified sequence; for example, as a segment of a
full-length cDNA or gene sequence, or the complete cDNA or gene sequence.

[0092] (b) As used herein, "comparison window" means includes reference to
a contiguous and specified segment of a polynucleotide sequence, wherein
the polynucleotide sequence may be compared to a reference sequence and
wherein the portion of the polynucleotide sequence in the comparison
window may comprise additions or deletions (i.e., gaps) compared to the
reference sequence (which does not comprise additions or deletions) for
optimal alignment of the two sequences. Generally, the comparison window
is at least 20 contiguous nucleotides in length, and optionally can be
30, 40, 50, 100, or longer. Those of skill in the art understand that to
avoid a high similarity to a reference sequence due to inclusion of gaps
in the polynucleotide sequence a gap penalty is typically introduced and
is subtracted from the number of matches.

[0093] Methods of alignment of sequences for comparison are well known in
the art. Optimal alignment of sequences for comparison may be conducted
by the local homology algorithm of Smith and Waterman. Adv. Appl. Math.
2: 482 (1981); by the homology alignment algorithm of Needleman and
Wunsch, J. Mol Biol 48: 443 (1970); by the search for similarity method
of Pearson and Lipman, Proc. Natl. Acad. Sci. 85: 2444 (1988); by
computerized implementations of these algorithms, including, but not
limited to: CLUSTAL in the PC/Gene program by Intelligenetics, Mountain
View, Calif., GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin
Genetics Software Package, Genetics Computer Group (GCG), 575 Science
Dr., Madison, Wis., USA; the CLUSTAL program is well described by Higgins
and Sharp, Gene 73: 237-244 (1988); Higgins and Sharp, CABIOS 5: 151-153
(1989); Corpet, et al., Nucleic Acids Research 16: 10881-90 (1988);
Huang, et al., Computer Applications in the Biosciences 8: 155-65 (1992),
and Pearson, et al., Methods in Molecular Biology 24: 307-331 (1994). The
BLAST family of programs which can be used for database similarity
searches includes: BLASTN for nucleotide query sequences against
nucleotide database sequences; BLASTX for nucleotide query sequences
against protein database sequences; BLASTP for protein query sequences
against protein database sequences; TBLASTN for protein query sequences
against nucleotide database sequences; and TBLASTX for nucleotide query
sequences against nucleotide database sequences. See, Current Protocols
in Molecular Biology, Chapter 19, Ausubel, et al., Eds., Greene
Publishing and Wiley-Interscience, New York (1995).

[0094] GAP uses the algorithm of Needleman and Wunsch (J Mol Biol 48:
443-453 (1970)) to find the alignment of two complete sequences that
maximizes the number of matches and minimizes the number of gaps. GAP
considers all possible alignments and gap positions and creates the
alignment with the largest number of matched bases and the fewest gaps.
It allows for the provision of a gap creation penalty and a gap extension
penalty in units of matched bases. GAP must make a profit of gap creation
penalty number of matches for each gap it inserts. If a gap extension
penalty greater than zero is chosen, GAP must, in addition, make a profit
for each gap inserted of the over the length of the gap times the gap
extension penalty. Default gap creation penalty values and gap extension
penalty values in Version 10 of the Wisconsin Genetics Software Package
are 8 and 2, respectively, for protein sequences. For nucleotide
sequences the default gap creation penalty is 50 while the default gap
extension penalty is 3. The gap creation and gap extension penalties can
be expressed as an integer selected from the group of integers consisting
of from 0 to 100. Thus, for example, the gap creation and gap extension
penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50,
60, or greater.

[0095] GAP presents one member of the family of best alignments. There may
be many members of this family, but no other member has a better quality.
GAP displays four figures of merit for alignments: Quality, Ratio,
Identity, and Similarity. The Quality is the metric maximized in order to
align the sequences. Ratio is the quality divided by the number of bases
in the shorter segment. Percent Identity is the percent of the symbols
that actually match. Percent Similarity is the percent of the symbols
that are similar. Symbols that are across from gaps are ignored. A
similarity is scored when the scoring matrix value for a pair of symbols
is greater than or equal to 0.50, the similarity threshold. The scoring
matrix used in Version 10 of the Wisconsin Genetics Software Package is
BLOSUM62 (see Henikoff and Henikoff, Proc Natl Acad Sci USA 89:10915).
Unless otherwise stated, sequence identity/similarity values provided
herein refer to the value obtained using the GAP version 10 of Wisconsin
Genetic Software Package using default parameters.

[0096] Comparisons of polynucleotide sequences that are of substantially
different lengths can be determined by a combination of percent identity
between the two sequences times the ratio of the coding region. In other
words, Relation=% Identity.times.Ratio of the coding region. For example,
if a first polynucleotide is 100% identical at the nucleotide level, but
only represents 30% of the coding region of the second polynucleotide,
then it is expressed as 30% related.

[0097] (c) As used herein, "sequence identity" or "identity" in the
context of two nucleic acid or polypeptide sequences includes reference
to the residues in the two sequences, which are the same when aligned for
maximum correspondence over a specified comparison window. When
percentage of sequence identity is used in reference to proteins it is
recognized that residue positions which are not identical often differ by
conservative amino acid substitutions, where amino acid residues are
substituted for other amino acid residues with similar chemical
properties (e.g. charge or hydrophobicity) and therefore do not change
the functional properties of the molecule. Where sequences differ in
conservative substitutions, the percent sequence identity may be adjusted
upwards to correct for the conservative nature of the substitution.
Sequences, which differ by such conservative substitutions, are said to
have "sequence similarity" or "similarity". Means for making this
adjustment are well known to those of skill in the art. Typically this
involves scoring a conservative substitution as a partial rather than a
full mismatch, thereby increasing the percentage sequence identity. Thus,
for example, where an identical amino acid is given a score of 1 and a
non-conservative substitution is given a score of zero, a conservative
substitution is given a score between zero and 1. The scoring of
conservative substitutions is calculated, e.g., according to the
algorithm of Meyers and Miller, Computer Applic. Biol. Sci., 4: 11-17
(1988) e.g., as implemented in the program PC/GENE (Intelligenetics,
Mountain View, Califormia, USA).

[0098] (d) As used herein, "percentage of sequence identity" means the
value determined by comparing two optimally aligned sequences over a
comparison window, wherein the portion of the polynucleotide sequence in
the comparison window may comprise additions or deletions (i.e., gaps) as
compared to the reference sequence (which does not comprise additions or
deletions) for optimal alignment of the two sequences. The percentage is
calculated by determining the number of positions at which the identical
nucleic acid base or amino acid residue occurs in both sequences to yield
the number of matched positions, dividing the number of matched positions
by the total number of positions in the window of comparison and
multiplying the result by 100 to yield the percentage of sequence
identity.

[0099] Nucleic Acids

[0100] The present invention provides, among other things, isolated
nucleic acids of RNA, DNA, and analogs and/or chimeras thereof,
comprising a polynucleotide of the present invention.

[0111] The present invention provides an isolated nucleic acid comprising
a polynucleotide of the present invention, wherein the polynucleotides
are amplified, under nucleic acid amplification conditions, from a plant
nucleic acid library. Nucleic acid amplification conditions for each of
the variety of amplification methods are well known to those of ordinary
skill in the art. The plant nucleic acid library can be constructed from
a monocot such as a cereal crop. Exemplary cereals include corn, sorghum,
alfalfa, canola, wheat, or rice. The plant nucleic acid library can also
be constructed from a dicot such as soybean. Zea mays lines B73, PHRE1,
A632, BMS-P2#10, W23, and Mol7 are known and publicly available. Other
publicly known and available maize lines can be obtained from the Maize
Genetics Cooperation (Urbana, Ill.). Wheat lines are available from the
Wheat Genetics Resource Center (Manhattan, Kans.).

[0112] The nucleic acid library may be a cDNA library, a genomic library,
or a library generally constructed from nuclear transcripts at any stage
of intron processing. cDNA libraries can be normalized to increase the
representation of relatively rare cDNAs. In optional embodiments, the
cDNA library is constructed using an enriched full-length cDNA synthesis
method. Examples of such methods include Oligo-Capping (Maruyama, K. and
Sugano, S. Gene 138: 171-174, 1994), Biotinylated CAP Trapper (Carninci,
et al. Genomics 37: 327-336, 1996), and CAP Retention Procedure (Edery,
E., Chu, L. L., et al. Molecular and Cellular Biology 15: 3363-3371,
1995). Rapidly growing tissues or rapidly dividing cells are preferred
for use as a mRNA source for construction of a cDNA library. Growth
stages of corn is described in "How a Corn Plant Develops," Special
Report No. 48, Iowa State University of Science and Technology
Cooperative Extension Service, Ames, Iowa, Reprinted February 1993.

[0113] A polynucleotide of this embodiment (or subsequences thereof) can
be obtained, for example, by using amplification primers which are
selectively hybridized and primer extended, under nucleic acid
amplification conditions, to at least two sites within a polynucleotide
of the present invention, or to two sites within the nucleic acid which
flank and comprise a polynucleotide of the present invention, or to a
site within a polynucleotide of the present invention and a site within
the nucleic acid which comprises it. Methods for obtaining 5' and/or 3'
ends of a vector insert are well known in the art. See, e.g., RACE (Rapid
Amplification of Complementary Ends) as described in Frohman, M. A., in
PCR Protocols: A Guide to Methods and Applications, M. A. Innis, D. H.
Gelfand, J. J. Sninsky, T. J. White, Eds. (Academic Press, Inc., San
Diego), pp. 28-38 (1990)); see also, U.S. Pat. No. 5,470,722, and Current
Protocols in Molecular Biology, Unit 15.6, Ausubel, et al., Eds., Greene
Publishing and Wiley-Interscience, New York (1995); Frohman and Martin,
Techniques 1:165 (1989).

[0114] Preferably, the primers are complementary to a subsequence of the
target nucleic acid which they amplify but may have a sequence identity
ranging from about 85% to 99% relative to the polynucleotide sequence
which they are designed to anneal to. As those skilled in the art will
appreciate, the sites to which the primer pairs will selectively
hybridize are chosen such that a single contiguous nucleic acid can be
formed under the desired nucleic acid amplification conditions. The
primer length in nucleotides is selected from the group of integers
consisting of from at least 15 to 50. Thus, the primers can be at least
15, 18, 20, 25, 30, 40, or 50 nucleotides in length. Those of skill will
recognize that a lengthened primer sequence can be employed to increase
specificity of binding (i.e., annealing) to a target sequence. A
non-annealing sequence at the 5' end of a primer (a "tail") can be added,
for example, to introduce a cloning site at the terminal ends of the
amplicon.

[0115] The amplification products can be translated using expression
systems well known to those of skill in the art. The resulting
translation products can be confirmed as polypeptides of the present
invention by, for example, assaying for the appropriate catalytic
activity (e.g., specific activity and/or substrate specificity), or
verifying the presence of one or more linear epitopes, which are specific
to a polypeptide of the present invention. Methods for protein synthesis
from PCR derived templates are known in the art and available
commercially. See, e.g., Amersham Life Sciences, Inc, Catalog '97, p.354.

[0116] C. Polynucleotides Which Selectively Hybridize to a Polynucleotide
of (A) or (B)

[0117] The present invention provides isolated nucleic acids comprising
polynucleotides of the present invention, wherein the polynucleotides
selectively hybridize, under selective hybridization conditions, to a
polynucleotide of section (A) or (B) as discussed above. Thus, the
polynucleotides of this embodiment can be used for isolating, detecting,
and/or quantifying nucleic acids comprising the polynucleotides of (A) or
(B). For example, polynucleotides of the present invention can be used to
identify, isolate, or amplify partial or full-length clones in a
deposited library. In some embodiments, the polynucleotides are genomic
or cDNA sequences isolated or otherwise complementary to a cDNA from a
dicot or monocot nucleic acid library. Exemplary species of monocots and
dicots include, but are not limited to: maize, canola, soybean, cotton,
wheat, sorghum, sunflower, alfalfa, oats, sugar cane, millet, barley, and
rice. The cDNA library comprises at least 50% to 95% full-length
sequences (for example, at least 50%, 60%, 70%, 80%, 90%, or 95%
full-length sequences). The cDNA libraries can be normalized to increase
the representation of rare sequences. See, e.g., U.S. Pat. No. 5,482,845.
Low stringency hybridization conditions are typically, but not
exclusively, employed with sequences having a reduced sequence identity
relative to complementary sequences. Moderate and high stringency
conditions can optionally be employed for sequences of greater identity.
Low stringency conditions allow selective hybridization of sequences
having about 70% to 80% sequence identity and can be employed to identify
orthologous or paralogous sequences.

[0118] D. Polynucleotides Having a Specific Sequence Identify with the
Polynucleotides of (A), (B) or (C)

[0119] The present invention provides isolated nucleic acids comprising
polynucleotides of the present invention, wherein the polynucleotides
have a specified identity at the nucleotide level to a polynucleotide as
disclosed above in sections (A), (B), or (C), above. The percentage of
identity to a reference sequence is at least 60% and, rounded upwards to
the nearest integer, can be expressed as an integer selected from the
group of integers consisting of from 60 to 99. Thus, for example, the
percentage of identity to a reference sequence can be at least 70%, 75%,
76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0120] Optionally, the polynucleotides of this embodiment will encode a
polypeptide that will share an epitope with a polypeptide encoded by the
polynucleotides of section (A), (B), or (C). Thus, these polynucleotides
encode a first polypeptide, which elicits production of antisera
comprising which are specifically reactive to a second polypeptide
encoded by a polynucleotide of (A), (B), or (C). However, the first
polypeptide does not bind to antisera raised against itself when the
antisera have been fully immunosorbed with the first polypeptide. Hence,
the polynucleotides of this embodiment can be used to generate antibodies
for use in, for example, the screening of expression libraries for
nucleic acids comprising polynucleotides of (A), (B), or (C), or for
purification of, or in immunoassays for, polypeptides encoded by the
polynucleotides of (A), (B), or (C). The polynucleotides of this
embodiment embrace nucleic acid sequences, which can be employed for
selective hybridization to a polynucleotide encoding a polypeptide of the
present invention.

[0121] Screening polypeptides for specific binding to antisera can be
conveniently achieved using peptide display libraries. This method
involves the screening of large collections of peptides for individual
members having the desired function or structure. Antibody screening of
peptide display libraries is well known in the art. The displayed peptide
sequences can be from 3 to 5000 or more amino acids in length, frequently
from 5-100 amino acids long, and often from about 8 to 15 amino acids
long. In addition to direct chemical synthetic methods for generating
peptide libraries, several recombinant DNA methods have been described.
One type involves the display of a peptide sequence on the surface of a
bacteriophage or cell. Each bacteriophage or cell contains the nucleotide
sequence encoding the particular displayed peptide sequence. Such methods
are described in PCT patent publication Nos. 91/17271, 91/18980,
91/19818, and 93/08278. Other systems for generating libraries of
peptides have aspects of both in vitro chemical synthesis and recombinant
methods. See PCT Patent publication Nos. 92/05258, 92/14843, and
96/19256. See also, U.S. Pat. Nos. 5,658,754; and 5,643,768. Peptide
display libraries, vectors, and screening kits are commercially available
from such suppliers as Invitrogen (Carlsbad, Calif.).

[0122] E. Polynucleotides Complementary to the Polynucleotides of (A)-(D).

[0123] The present invention provides isolated nucleic acids comprising
polynucleotides complementary to the polynucleotides of paragraphs A-D,
above. As those of skill in the art will recognize, complementary
sequences base-pair throughout the entirety of their length with the
polynucleotides of sections (A)-(D) (i.e., have 100% sequence identity
over their entire length.) Complementary bases associate through hydrogen
bonding in double stranded nucleic acids. For example, the following base
pairs are complementary: guanine and cytosine; adenine and thymine; and
adenine and uracil.

[0124] F. Polynucleotides That are Subsequences of the Polynucleotides of
(A)-(E)

[0126] The subsequences of the present invention can comprise structural
characteristics of the sequence from which it is derived. Alternatively,
the subsequences can lack certain structural characteristics of the
larger sequence from which it is derived such as poly (A) tail.
Optionally, a subsequence from a polynucleotide encoding a polypeptide
having at least one linear epitope in common with a prototype polypeptide
sequence as provided in (a), above, may encode an epitope in common with
the prototype sequence. Alternatively, the subsequence may not encode an
epitope in common with the prototype sequence but can be used to isolate
the larger sequence by, for example, nucleic acid hybridization with the
sequence from which it's derived. Subsequences can be used to modulate or
detect gene expression by introducing into the subsequence compounds,
which bind, intercalate, cleave and/or crosslink to nucleic acids.
Exemplary compounds include acridine, psoralen, phenanthroline,
naphthoquinone, daunomycin, or chloroethylaminoaryl conjugates. In
addition, by virtue of the fact that WRKY polynucleotides contain DNA
binding regions, such as the TGAC-containing W box, subsequences of a
WRKY polynucleotide could be used to test the binding of target DNA or to
identify genes or promoters that respond to the WRKY domains.

[0127] Construction of Nucleic Acids

[0128] The isolated nucleic acids of the present invention can be made
using (a) standard recombinant methods, (b) synthetic techniques, or
combinations thereof. In some embodiments, the polynucleotides of the
present invention will be cloned, amplified, or otherwise constructed
from a monocot. In preferred embodiments the monocot is Zea mays.

[0129] The nucleic acids may conveniently comprise sequences in addition
to a polynucleotide of the present invention. For example, a
multi-cloning site comprising one or more endonuclease restriction sites
may be inserted into the nucleic acid to aid in isolation of the
polynucleotide. Also, translatable sequences may be inserted to aid in
the isolation of the translated polynucleotide of the present invention.
For example, a hexa-histidine marker sequence provides a convenient means
to purify the proteins of the present invention. A polynucleotide of the
present invention can be attached to a vector, adapter, or linker for
cloning and/or expression of a polynucleotide of the present invention.
Additional sequences may be added to such cloning and/or expression
sequences to optimize their function in cloning and/or expression, to aid
in isolation of the polynucleotide, or to improve the introduction of the
polynucleotide into a cell. Typically, the length of a nucleic acid of
the present invention less the length of its polynucleotide of the
present invention is less than 20 kilobase pairs, often less than 15 kb,
and frequently less than 10 kb. Use of cloning vectors, expression
vectors, adapters, and linkers is well known and extensively described in
the art. For a description of various nucleic acids see, for example,
Stratagene Cloning Systems, Catalogs 1999 (La Jolla, Calif.); and,
Amersham Life Sciences, Inc, Catalog '99 (Arlington Heights, Ill.).

[0130] A. Recombinant Methods for Constructing Nucleic Acids

[0131] The isolated nucleic acid compositions of this invention, such as
RNA, cDNA, genomic DNA, or a hybrid thereof, can be obtained from plant
biological sources using any number of cloning methodologies known to
those of skill in the art. In some embodiments, oligonucleotide probes
that selectively hybridize, under stringent conditions, to the
polynucleotides of the present invention are used to identify the desired
sequence in a cDNA or genomic DNA library. Isolation of RNA and
construction of cDNA and genomic libraries is well known to those of
ordinary skill in the art. See, e.g., Plant Molecular Biology: A
Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997); and,
Current Protocols in Molecular Biology, Ausubel, et al., Eds., Greene
Publishing and Wiley-Interscience, New York (1995).

[0132] A1. Full-length Enriched cDNA Libraries

[0133] A number of cDNA synthesis protocols have been described which
provide enriched full-length cDNA libraries. Enriched full-length cDNA
libraries are constructed to comprise at least 60%, and more preferably
at least 70%, 80%, 90% or 95% full-length inserts amongst clones
containing inserts. The length of insert in such libraries can be at
least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more kilobase pairs. Vectors to
accommodate inserts of these sizes are known in the art and available
commercially. See, e.g., Stratagene's lambda ZAP Express (cDNA cloning
vector with 0 to 12 kb cloning capacity). An exemplary method of
constructing a greater than 95% pure full-length cDNA library is
described by Carninci et al., Genomics, 37:327-336 (1996). Other methods
for producing full-length libraries are known in the art. See, e.g.,
Edery et al., Mol. Cell Biol., 15(6):3363-3371 (1995); and, PCT
Application WO 96/34981.

[0134] A2 Normalized or Subtracted cDNA Libraries

[0135] A non-normalized cDNA library represents the mRNA population of the
tissue it was made from. Since unique clones are out-numbered by clones
derived from highly expressed genes their isolation can be laborious.
Normalization of a cDNA library is the process of creating a library in
which each clone is more equally represented. Construction of normalized
libraries is described in Ko, Nucl Acids Res, 18(19):5705-5711 (1990);
Patanjali et al., Proc. Natl. Acad. U.S.A., 88:1943-1947 (1991); U.S.
Pat. Nos. 5,482,685, 5,482,845, and 5,637,685. In an exemplary method
described by Soares et al., normalization resulted in reduction of the
abundance of clones from a range of four orders of magnitude to a narrow
range of only 1 order of magnitude. Proc. Natl. Acad. Sci. USA,
91:9228-9232 (1994).

[0137] To construct genomic libraries, large segments of genomic DNA are
generated by fragmentation, e.g. using restriction endonucleases, and are
ligated with vector DNA to form concatemers that can be packaged into the
appropriate vector. Methodologies to accomplish these ends and sequencing
methods to verify the sequence of nucleic acids are well known in the
art. Examples of appropriate molecular biological techniques and
instructions sufficient to direct persons of skill through many
construction, cloning, and screening methodologies are found in Sambrook,
et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring
Harbor Laboratory Vols. 1-3 (1989), Methods in Enzymology, Vol. 152:
Guide to Molecular Cloning Techniques, Berger and Kimmel, Eds., San
Diego: Academic Press, Inc. (1987), Current Protocols in Molecular
Biology, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience,
New York (1995); Plant Molecular Biology: A Laboratory Manual, Clark,
Ed., Springer-Verlag, Berlin (1997). Kits for construction of genomic
libraries are also commercially available.

[0138] The cDNA or genomic library can be screened using a probe based
upon the sequence of a polynucleotide of the present invention such as
those disclosed herein. Probes may be used to hybridize with genomic DNA
or cDNA sequences to isolate homologous genes in the same or different
plant species. Those of skill in the art will appreciate that various
degrees of stringency of hybridization can be employed in the assay; and
either the hybridization or the wash medium can be stringent.

[0139] The nucleic acids of interest can also be amplified from nucleic
acid samples using amplification techniques. For instance, polymerase
chain reaction (PCR) technology can be used to amplify the sequences of
polynucleotides of the present invention and related genes directly from
genomic DNA or cDNA libraries. PCR and other in vitro amplification
methods may also be useful, for example, to clone nucleic acid sequences
that code for proteins to be expressed, to make nucleic acids to use as
probes for detecting the presence of the desired mRNA in samples, for
nucleic acid sequencing, or for other purposes. The T4 gene 32 protein
(Boehringer Mannheim) can be used to improve yield of long PCR products.

[0140] PCR-based screening methods have been described. Wilfinger et al.
describe a PCR-based method in which the longest cDNA is identified in
the first step so that incomplete clones can be eliminated from study.
BioTechniques, 22(3): 481-486 (1997). Such methods are particularly
effective in combination with a full-length cDNA construction
methodology, above.

[0141] B. Synthetic Methods for Constructing Nucleic Acids

[0142] The isolated nucleic acids of the present invention can also be
prepared by direct chemical synthesis by methods such as the
phosphotriester method of Narang et al., Meth. Enzymol. 68: 90-99 (1979);
the phosphodiester method of Brown et al., Meth. Enzymol. 68: 109-151
(1979); the diethylphosphoramidite method of Beaucage et al., Tetra.
Lett. 22: 1859-1862 (1981); the solid phase phosphoramidite triester
method described by Beaucage and Caruthers, Tetra. Letts. 22(20):
1859-1862 (1981), e.g., using an automated synthesizer, e.g., as
described in Needham-VanDevanter et al., Nucleic Acids Res., 12:
6159-6168 (1984); and, the solid support method of U.S. Pat. No.
4,458,066. Chemical synthesis generally produces a single stranded
oligonucleotide. This may be converted into double stranded DNA by
hybridization with a complementary sequence, or by polymerization with a
DNA polymerase using the single strand as a template. One of skill will
recognize that while chemical synthesis of DNA is best employed for
sequences of about 100 bases or less, longer sequences may be obtained by
the ligation of shorter sequences.

[0143] Recombinant Expression Cassettes

[0144] The present invention further provides recombinant expression
cassettes comprising a nucleic acid of the present invention. A nucleic
acid sequence coding for the desired polynucleotide of the present
invention, for example a cDNA or a genomic sequence encoding a full
length polypeptide of the present invention, can be used to construct a
recombinant expression cassette which can be introduced into the desired
host cell. A recombinant expression cassette will typically comprise a
polynucleotide of the present invention operably linked to
transcriptional initiation regulatory sequences which will direct the
transcription of the polynucleotide in the intended host cell, such as
tissues of a transformed plant.

[0145] For example, plant expression vectors may include (1) a cloned
plant gene under the transcriptional control of 5' and 3' regulatory
sequences and (2) a dominant selectable marker. Such plan expression
vectors may also contain, if desired, a promoter regulatory region (e.g.,
one conferring inducible or constitutive, environmentally- or
developmentally-regulated, or cell- or tissue-specific/selective
expression), a transcription initiation start site, a ribosome binding
site, an RNA processing signal, a transcription termination site, and/or
a polyadenylation signal.

[0146] A number of promoters can be used in the practice of the invention.
A plant promoter fragment can be employed which will direct expression of
a polynucleotide of the present invention in all tissues of a regenerated
plant. Such promoters are referred to herein as "constitutive" promoters
and are active under most environmental conditions and stated of
development or cell differentiation. Examples of constitutive promoters
include the cauliflower mosaic virus (CaMV) 35S transcription initiation
region, the 1'- or 2'-promoter derived from T-DNA of Agrobacterium
tumefaciens, the ubiquitin 1 promoter (Christensen, et al. Plant Mol Biol
18, 675-689 (1992); Bruce, et al., Proc Natl Acad Sci USA 86, 9692-9696
(1989)), the Smas promoter, the cinnamyl alcohol dehydrogenase promoter
(U.S. Pat. No, 5,683,439), the Nos promoter, the pEmu promoter, the
rubisco promoter, the GRP 1-8 promoter, the maize constitutive promoters
described in PCT Publication No. WO 99/43797 which include the histone
H2B, metallothionein, alpha-tubulin 3, elongation factor efla, ribosomal
protein rps8, chlorophyll a/b binding protein, and
glyceraldehyde-3-phosphate dehydrogenase promoters, and other
transcription initiation regions from various plant genes known to those
of skill. The preferred promoter is a pathogen-inducible promoter such as
the Sclerotinia-inducible promoters PR5-2 and BAP, which can be found in
co-pending U.S. application number 09/185,292, filed Oct. 10, 2000.
Another preferred inducible promoter is a promoter designed with the
estrogen response element (ERE) (Klein-Hitpass, et al., Nuc. Acids Res.
16:647-63 (1988)). For example, four repeats of the ERE element are fused
upstream of the Adhl minimal promoter, which is fused upstream of the
Adhl intron.

[0147] Where low level expression is desired, weak promoters will be used.
It is recognized that weak inducible promoters may be used. Additionally,
either a weak constitutive or a weak tissue specific promoter may be
used. Generally, by a "weak promoter" is intended a promoter that drives
expression of a coding sequence at a low level. By low level is intended
at levels of about {fraction (1/1000)} transcripts to about {fraction
(1/100,000)} transcripts to about {fraction (1/500,000)} transcripts.
Alternatively, it is recognized that weak promoters also encompass
promoters that are expresses in only a few cells and not in others to
give a total low level of expression. Such weak constitutive promoters
include, for example, the core promoter of the Rsyn7 (WO 97/44756), the
core 35S CaMV promoter, and the like. Where a promoter is expressed at
unacceptably high levels, portions of the promoter sequence can be
deleted or modified to decrease expression levels. Additionally, to
obtain a varied series in the level of expression, one can also make a
set of transgenic plants containing the polynucleotides of the present
invention with a strong constitutive promoter, and then rank the
transgenic plants according to the observed level of expression. The
transgenic plants will show a variety in performance, from high
expression to low expression. Factors such as chromosomal position
effect, cosuppression, and the like will affect the expression of the
polynucleotide.

[0148] Alternatively, the plant promoter can direct expression of a
polynucleotide of the present invention under environmental control. Such
promoters are referred to here as "inducible" promoters. Environmental
conditions that may effect transcription by inducible promoters include
pathogen attack, anaerobic conditions, or the presence of light. Examples
of inducible promoters are the Adhl promoter, which is inducible by
hypoxia or cold stress, the Hsp7O promoter, which is inducible by heat
stress, and the PPDK promoter, which is inducible by light. Examples of
pathogen-inducible promoters include those from proteins, which are
induced following infection by a pathogen; e.g., PR proteins, SAR
proteins, beta-a,3-glucanase, chitinase, etc. See, for example, Redolfi,
et al., Meth J. Plant Pathol. 89:245-254 (1983); Uknes et al., The Plant
Cell 4:645-656 (1992); Van Loon, Plant Mol. Virol. 4:111-116 (1985); and
PCT Publication No. WO 99/43819.

[0151] Examples of promoters under developmental control include promoters
that initiate transcription only, or preferentially, in certain tissues,
such as leaves, roots, fruit, seeds, or flowers. Exemplary promoters
include the anther specific promoter 5126 (U.S. Pat. Nos. 5,689,049 and
5,689,051), glob-1 promoter, and gamma-zein promoter. An exemplary
promoter for leaf- and stalk-preferred expression is MS8-15 (WO
98/00533). Examples of seed-preferred promoters included, but are not
limited to, 27 kD gamma zein promoter and waxy promoter (Boronat, et al.,
Plant Sci, 47:95-102 (1986); Reina, et al., Nucleic Acids Res 18(21):6426
(1990); and Kloesgen, et al., Mol Gen Genet 203:237-244 (1986)).
Promoters that express in the embryo, pericarp, and endosperm are
disclosed in PCT Publication WO 00/11177, published on Mar. 2, 2000, and
PCT Publication WO 00/12733, published on Mar. 9, 2000, both of which are
hereby incorporated by reference. The operation of a promoter may also
vary depending on its location in the genome. Thus, a developmentally
regulated promoter may become fully or partially constitutive in certain
locations. A developmentally regulated promoter can also be modified, if
necessary, for weak expression.

[0152] Both heterologous and non-heterologous (i.e. endogenous) promoters
can be employed to direct expression of the nucleic acids of the present
invention. These promoters can also be used, for example, in recombinant
expression cassettes to drive expression of antisense nucleic acids to
reduce, increase, or alter concentration and/or composition of the
proteins of the present invention in a desired tissue. Thus, in some
embodiments, the nucleic acid construct will comprise a promoter
functional in a plant cell, such as in Zea Mays, operably linked to a
polynucleotide of the present invention. Promoters useful in these
embodiments include the endogenous promoters driving expression of a
polypeptide of the present invention.

[0153] In some embodiments, isolated nucleic acids which serve as a
promoter or enhancer elements can be introduced in the appropriate
position (generally upstream) of a non-heterologous form of a
polynucleotide of the present invention so as to up or down regulate
expression of a polynucleotide of the present invention. For example,
endogenous promoters can be altered in vivo by mutation, deletion, and/or
substitution (see, Kmiec, U.S. Pat. No. 5,565,350; Zarling et al.,
PCT/US93?03868), or isolated promoters can be introduced into a plant
cell in the proper orientation and distance from a gene of the present
invention so as to control the expression of the gene. Gene expression
can be modulated under conditions suitable for plant growth so as to
alter the total concentration and/or alter the composition of the
polypeptides of the present invention in plant cell. Thus, the present
invention provides compositions, and methods for making, heterologous
promoters and/or enhancers operably linked to a native, endogenous (i.e.,
non-heterologous) form of a polynucleotide of the present invention.

[0154] If polypeptide expression is desired, it is generally desirable to
include a polyadenylation region at the 3'-end of a polynucleotide coding
region. The polyadenylation region can be derived from the natural gene,
from a variety of other plant genes, or from T-DNA. The 3' end sequence
to be added can be derived from, for example, the nopaline synthase or
octopine synthase genes, or alternatively from another plant gene, or
less preferably from any other eukaryotic gene.

[0155] An intron sequence can be added to the 5' untranslated region or
the coding sequence of the partial coding sequence to increase the amount
of the mature message that accumulates in the cytosol. Inclusion of a
spliceable intron in the transcription unit in both plant and animal
expression constructs has been shown to increase gene expression at both
the mRNA and protein levels up to 1000-fold, Buchman and Berg, Mol. Cell
Biol. 8: 4395-4405 (1988); Callis et al., Genes Dev. 1: 1183-1200 (1987).
Such intron enhancement of gene expression is typically greatest when
placed near the 5' end of the transcription unit. Use of the maize
introns Adh1-S intron 1, 2, and 6, the Bronze-1 intron are known in the
art. See generally, The Maize Handbook, Chapter 116, Freeling and Walbot,
Eds., Springer, N.Y. (1994).

[0156] The vector comprising the sequences from a polynucleotide of the
present invention will typically comprise a marker gene, which confers a
selectable phenotype on plant cells. Usually, the selectable marker gene
will encode antibiotic resistance, with suitable genes including genes
coding for resistance to the antibiotic spectinomycin (e.g., the aada
gene), the streptomycin phosphotransferase (SPT) gene coding for
streptomycin resistance, the neomycin phosphotransferase (NPTII) gene
encoding kanamycin or geneticin resistance, the hygromycin
phosphotransferase (HPT) gene coding for hygromycin resistance, genes
coding for resistance to herbicides which act to inhibit the action of
acetolactate synthase (ALS), in particular the sulfonylurea-type
herbicides (e.g., the acetolactate synthase (ALS) gene containing
mutations leading to such resistance in particular the S4 and/or Hra
mutations), genes coding for resistance to herbicides which act to
inhibit action of glutamine synthase, such as phosphinothricin or basta
(e.g., the bar gene), or other such genes known in the art. The bar gene
encodes resistance to the herbicide basta, the nptII gene encodes
resistance to the antibiotics kanamycin and geneticin, and the ALS gene
encodes resistance to the herbicide chlorsulfuron.

[0157] Typical vectors useful for expression of genes in higher plants are
well known in the art and include vectors derived from the tumor-induced
(Ti) plasmid of Agrobacterium tumefaciens described by Rogers et al.,
Meth. In Enzymol., 153:253-277 (1987). These vectors are plant
integrating vectors in that upon transformation, the vectors integrate a
portion of vector DNA into the genome of the host plant. Exemplary A.
tumefaciens vectors useful herein are plasmids pKYLX6 and pKYLX7 of
Schardl et al., Gene, 61:1-11(1987) and Berger et al., Proc. Natl. Acad.
Sci. U.S.A., 86:8402-8406 (1989). Another useful vector herein is plasmid
pBI101.2 that is available from Clontech Laboratories, Inc. (Palo Alto,
Calif.).

[0158] A polynucleotide of the present invention can be expressed in
either sense or anti-sense orientation as desired. It will be appreciated
that control of gene expression in either sense or anti-sense orientation
can have a direct impact on the observable plant characteristics.
Antisense technology can be conveniently used to inhibit gene expression
in plants. To accomplish this, a nucleic acid segment from the desired
gene is cloned and operably linked to a promoter such that the anti-sense
strand of RNA will be transcribed. The construct is then transformed into
plants and the antisense strand of RNA is produced. In plant cells, it
has been shown that antisense RNA inhibits gene expression by preventing
the accumulation of mRNA which encodes the enzyme of interest, see, e.g.,
Sheehy et al., Proc. Nat'l. Acad. Sci (USA) 85:8805-8809 (1988); and
Hiatt et al., U.S. Pat. No. 4,801,340.

[0159] Another method of suppression is sense suppression. Introduction of
nucleic acid configured in the sense orientation has been shown to be an
effective means by which to block the transcription of target genes. For
an example of the use of this method to modulate expression of endogenous
genes see, Napoli et al., The Plant Cell 2:279-289 (1990) and U.S. Pat.
No. 5,034,323.

[0160] Catalytic RNA molecules or ribozymes can also be used to inhibit
expression of plant genes. It is possible to design ribozymes that
specifically pair with virtually any target RNA and cleave the
phosphodiester backbone at a specific location, thereby functionally
inactivating the target RNA. In carrying out this cleavage, the ribozyme
is not itself altered, and is thus capable of recycling and cleaving
other molecules, making it a true enzyme. The inclusion of ribozyme
sequences within antisense RNAs confers RNA-cleaving activity upon them,
thereby increasing the activity of the constructs. The design and use of
target RNA-specific ribozymes is described in Haseloff et al., Nature
334:585-591 (1988).

[0161] A variety of cross-linking agents, alkylating agents and radical
generating species as pendant groups on polynucleotides of the present
invention can be used to bind, label, detect, and/or cleave nucleic
acids. For example, Vlassov, V. V., et al., Nucleic Acids Res (1986)
14:4065-4076, describe covalent bonding of a single-stranded DNA fragment
with alkylating derivatives of nucleotides complementary to target
sequences. A report of similar work by the same group is that by Knorre,
D. G., et al., Biochimie (1985) 67:785-789. Iverson and Dervan also
showed sequence-specific cleavage of single-stranded DNA meditated by
incorporation of a modified nucleotide which was capable of activating
cleavage (J Am Chem Soc (1987) 109:1241-1243). Meyer, R. B. et al., J Am
Chem Soc (1989) 111:8517-8519, effect covalent crosslinking to a target
nucleotide using an alkylating agent complementary to the single-stranded
target nucleotide sequence. A photoactivated crosslinking to
single-stranded oligonucleotides meditated by psoralen was disclosed by
Lee, B. L., et al., Biochemistry (1988) 27:3197-3203. Use of crosslinking
in triple-helix forming probes was also disclosed by Home et al., J. Am
Chem Soc (1990) 112:2435-2437. Use of N4, N4-ethanocytosine as an
alkylating agent to crosslink to single-stranded oligonucleotides has
also been described by Webb and Matteucci, J Am Chem Soc (1986)
108:2764-2765; Nucleic Acids Res (1986) 14:7661-7674; Feteritz et al., J.
Am. Chem. Soc. 113:4000 (1991). Various compounds to bind, detect, label,
and/or cleave nucleic acids are known in the art. See, for example, U.S.
Pat. Nos. 5,543,507; 5,672,593; 5,484,908; 5,256,648; and 5,681,941.

[0162] Proteins

[0163] The isolated proteins of the present invention comprise a
polypeptide having at least 10 amino acids encoded by any one of the
polynucleotides of the present invention as discussed more fully, above,
or polypeptides which are conservatively modified variants thereof. The
proteins of the present invention or variants thereof can comprise any
number of contiguous amino acid residues from a polypeptide of the
present invention, wherein that number is selected from the group of
integers consisting of from 10 to the number of residues in a full-length
polypeptide of the present invention. Optionally, this subsequence of
contiguous amino acids is at least 15, 20, 25, 30, 35, or 40 amino acids
in length, often at least 50, 60, 70, 80, or 90 amino acids in length.
Further, the number of such subsequences can be any integer selected from
the group consisting of from 1 to 20, such as 2, 3, 4, or 5.

[0164] As those of skill will appreciate, the present invention includes
catalytically active polypeptides of the present invention (i.e.,
enzymes). Catalytically active polypeptides have a specific activity of
at least 20%, 30%, or 40%, and preferably at least 50%, 60%, or 70%, and
most preferably at least 80%, 90%, or 95% that of the native
(non-synthetic), endogenous polypeptide. Further, the substrate
specificity (k.sub.cat/K.sub.m) is optionally substantially similar to
the native (non-synthetic), endogenous polypeptide. Typically, the
K.sub.m will be at least 30%, 40%, or 50%, that of the native
(non-synthetic), endogenous polypeptide; and more preferably at least
60%, 70%, 80%, or 90%. Methods of assaying and quantifying measures of
enzymatic activity and substrate specificity (k.sub.cat/K.sub.m), are
well known to those of skill in the art.

[0165] Generally, the proteins of the present invention will, when
presented as an immunogen, elicit production of an antibody specifically
reactive to a polypeptide of the present invention. Further, the proteins
of the present invention will not bind to antisera raised against a
polypeptide of the present invention, which has been fully immunosorbed
with the same polypeptide. Immunoassays for determining binding are well
known to those of skill in the art. A preferred immunoassay is a
competitive immunoassay as discussed, infra. Thus, the proteins of the
present invention can be employed as immunogens for constructing
antibodies immunoreactive to a protein of the present invention for such
exemplary utilities as immunoassays or protein purification techniques.

[0166] Expression of Proteins in Host Cells

[0167] Using the nucleic acids of the present invention, one may express a
protein of the present invention in a recombinantly engineered cell such
as bacteria, yeast, insect, mammalian, or preferably plant cells. The
cells produce the protein in a non-natural condition. (e.g., in quantity,
composition, location, and/or time), because they have been genetically
altered through human intervention to do so.

[0168] It is expected that those of skill in the art are knowledgeable in
the numerous expression systems available for expression of a nucleic
acid encoding a protein of the present invention. No attempt to describe
in detail the various methods known for the expression of proteins in
prokaryotes or eukaryotes will be made.

[0169] In brief summary, the expression of isolated nucleic acids encoding
a protein of the present invention will typically be achieved by operably
linking, for example, the DNA or cDNA to a promoter (which is either
constitutive or regulatable), followed by incorporation into an
expression vector. The vectors can be suitable for replication and
integration in either prokaryotes or eukaryotes. Typical expression
vectors contain transcription and translation terminators, initiation
sequences, and promoters useful for regulation of the expression of the
DNA encoding a protein of the present invention. To obtain high level
expression of a cloned gene, it is desirable to construct expression
vectors which contain, at the minimum, a strong promoter to direct
transcription, a ribosome binding site for translational initiation, and
a transcription/translation terminator. One of skill would recognize that
modifications could be made to a protein of the present invention without
diminishing its biological activity. Some modifications may be made to
facilitate the cloning, expression, or incorporation of the targeting
molecule into a fusion protein. Such modifications are well known to
those of skill in the art and include, for example, a methionine added at
the amino terminus to provide an initiation site, or additional amino
acids (e.g., poly His) placed on either terminus to create conveniently
located purification sequences. Restriction sites or termination codons
can also be introduced.

[0170] A. Expression in Prokaryotes

[0171] Prokaryotic cells may be used as hosts for expression. Prokaryotes
most frequently are represented by various strains of E. coli; however,
other microbial strains may also be used. Commonly used prokaryotic
control sequences which are defined herein to include promoters for
transcription initiation, optionally with an operator, along with
ribosome binding sequences, include such commonly used promoters as the
beta lactamase (penicillinase) and lactose (lac) promoter systems (Chang
et al., Nature 198:1056 (1977)), the tryptophan (trp) promoter system
(Goeddel et al., Nucleic Acids Res. 8:4057 (1980)) and the lambda derived
P L promoter and N-gene ribosome binding site (Shimatake et al., Nature
292:128(1981)). The inclusion of selection markers in DNA vectors
transfected in E coli. is also useful. Examples of such markers include
genes specifying resistance to ampicillin, tetracycline, or
chloramphenicol.

[0172] The vector is selected to allow introduction into the appropriate
host cell. Bacterial vectors are typically of plasmid or phage origin.
Appropriate bacterial cells are infected with phage vector particles or
transfected with naked phage vector DNA. If a plasmid vector is used, the
bacterial cells are transfected with the plasmid vector DNA. Expression
systems for expressing a protein of the present invention are available
using Bacillus sp. and Salmonella (Palva et al., Gene 22: 229-235 (1983);
Mosbach, et al., Nature 302:543-545 (1983)).

[0173] B. Expression in Eukaryotes

[0174] A variety of eukaryotic expression systems such as yeast, insect
cell lines, plant and mammalian cells, are known to those of skill in the
art. As explained briefly below, a polynucleotide of the present
invention can be expressed in these eukaryotic systems. In some
embodiments, transformed/transfected plant cells, as discussed infra, are
employed as expression systems for production of the proteins of the
instant invention.

[0175] Synthesis of heterologous proteins in yeast is well known. Sherman,
F., et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory
(1982) is a well recognized work describing the various methods available
to produce the protein in yeast. Two widely utilized yeasts for
production of eukaryotic proteins are Saccharomyces cerevisiae and Pichia
pastoris. Vectors, strains, and protocols for expression in Saccharomyces
and Pichia are known in the art and available from commercial suppliers
(e.g., Invitrogen). Suitable vectors usually have expression control
sequences, such as promoters, including 3-phosphoglycerate kinase or
alcohol oxidase, and an origin of replication, termination sequences and
the like as desired.

[0176] A protein of the present invention, once expressed, can be isolated
from yeast by lysine the cells and applying standard protein isolation
techniques to the lists. The monitoring of the purification process can
be accomplished by using Western blot techniques or radioimmunoassay of
other standard immunoassay techniques.

[0177] The sequences encoding proteins of the present invention can also
be ligated to various expression vectors for use in transfecting cell
cultures of, for instance, mammalian, insect, or plant origin.
Illustrative cell cultures useful for the production of the peptides are
mammalian cells. Mammalian cell systems often will be in the form of
minelayers of cells although mammalian cell suspensions may also be used.
A number of suitable host cell lines capable of expressing intact
proteins have been developed in the art, and include the HEK293, BHK21,
and CHO cell lines. Expression vectors for these cells can include
expression control sequences, such as an origin of replication, a
promoter (e.g. the CMV promoter, a HSV tk promoter or pgk
(phosphoglycerate kinase) promoter), an enhancer (Queen et al., Immunol.
Rev. 89:49 (1986)), and necessary processing information sites, such as
ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., an
SV40 large T Ag poly A addition site), and transcriptional terminator
sequences. Other animal cells useful for production of proteins of the
present invention are available, for instance, from the American Type
Culture Collection.

[0179] As with yeast, when higher animal or plant host cells are employed,
polyadenylation or transcription terminator sequences are typically
incorporated into the vector. An example of a terminator sequence is the
polyadenylation sequence from the bovine growth hormone gene. Sequences
for accurate splicing of the transcript may also be included. An example
of a splicing sequence is the VP 1 intron from SV40 (Sprague, et al., J.
Virol. 45:773-781 (1983)). Additionally, gene sequences to control
replication in the host cell may be incorporated into the vector such as
those found in bovine papilloma virus type-vectors. Saveria-Campo, M.,
Bovine Papilloma Virus DNA a Eukaryotic Cloning Vector in DNA Cloning
Vol. II a Practical Approach, D. M. Glover, Ed., IRL Press, Arlington,
Virginia pp. 213-238(1985).

[0180] Transfection/Transformation of Cells

[0181] The method of transfortnation/transfection is not critical to the
instant invention; various methods of transformation or transfection are
currently available. As newer methods are available to transform crops or
other host cells they may be directly applied. Accordingly, a wide
variety of methods have been developed to insert a DNA sequence into the
genome of a host cell to obtain the transcription and/or translation of
the sequence to effect phenotypic changes in the organism. Thus, any
method, which provides for effective transformation/transfection may be
employed.

[0184] The cells, which have been transformed, may be grown into plants in
accordance with conventional ways. See, for example, McCormick et al.
(1986) Plant Cell Reports, 5:81-84. These plants may then be grown, and
either pollinated with the same transformed strain or different strains,
and the resulting hybrid having the desired phenotypic characteristic
identified. Two or more generations may be grown to ensure that the
subject phenotypic characteristics is stably maintained and inherited and
then seeds harvested to ensure the desired phenotype or other property
has been achieved. One of skill will recognize that after the recombinant
expression cassette is stably incorporated in transgenic plants and
confirmed to be operable, it can be introduced into other plants by
sexual crossing. Any of number of standard breeding techniques can be
used, depending upon the species to be crossed.

[0185] In vegetatively propagated crops, mature transgenic plants can be
propagated by the taking of cuttings or by tissue culture techniques to
produce multiple identical plants. Selection of desirable transgenics is
made and new varieties are obtained and propagated vegetatively for
commercial use. In seed propagated crops, mature transgenic plants can be
self crossed to produce a homozygous inbred plant. The inbred plant
produces seed containing the newly introduced heterologous nucleic acid.
These seeds can be grown to produce plans that would produce the selected
phenotype.

[0186] Parts obtained from the regenerated plant, such as flowers, seeds,
leaves, branches, fruit, and the like are included in the invention,
provided that these parts comprise cells comprising the isolated nucleic
acid of the present invention. Progeny and variants, and mutants of the
regenerated plants are also included within the scope of the invention,
provided that these parts comprise the introduced nucleic acid sequences.

[0187] A preferred embodiment is a transgenic plant that is homozygous for
the added heterologous nucleic acid; i.e., a transgenic plant that
contains two added nucleic acid sequences, one gene at the same locus on
each chromosome of a chromosome pair. A homozygous transgenic plant can
be obtained by sexually mating(selling) a heterozygous transgenic plant
that contains a single added heterologous nucleic acid, germinating some
of the seed produced and analyzing the resulting plants produced for
altered expression of a polynucleotide of the present invention relative
to a control plant (i.e., native, non-transgenic). Backcrossing to a
parental plant and out-crossing with a non-transgenic plant are also
contemplated.

[0189] Animal and lower eukaryotic (e.g., yeast) host cells are competent
or rendered competent for transfection by various means. There are
several well-known methods of introducing DNA into animal cells. These
include: calcium phosphate precipitation, fusion of the recipient cells
with bacterial protoplasts containing the DNA, treatment of the recipient
cells with liposomes containing the DNA, DEAE dextrin, electroporation,
biolistics, and micro-injection of the DNA directly into the cells. The
transfected cells are cultured by means well known in the art. Kuchler,
R. J., Biochemical Methods in Cell Culture and Virology, Dowden,
Hutchinson and Ross, Inc (1997).

[0190] The WRKY Transcriptional Regulatory Region

[0191] The transcriptional region for WRKY genes may be generally isolated
from the 5' untranslated region flanking their respective transcription
initiation sites. Methods for isolation of transcriptional regulatory
regions are well known in the art. By "isolated" is intended that the
transcriptional regulatory region sequences have been determined and can
be extracted by molecular techniques or synthesized by chemical means. In
either instance, the transcriptional regulatory region is removed from at
least one of its flanking sequences in its native state. The sequence for
the transcriptional regulatory region of sunflower WRKY1-2 can be found
in SEQ ID NO: 35.

[0192] It is recognized that regions in addition to the transcriptional
regulatory region may be used to initiate transcription. Such regions
include the UTR and even portions of the coding sequence particularly 5'
portions of the coding region. Generally, from about 3 nucleotides (1
codon) up to about 150 nucleotides (50 codons) of the 5' coding region
can be used. See, for example, McElroy et al. (1991) Mol Gen. Genet. 231:
150-160 and herein incorporated by reference, where expression vectors
were constructed based on the rice actin 1 5' region.

[0193] Comparable transcriptional regulatory regions from other plants may
be obtained by utilization of the coding or promoter sequences of the
invention. Using the WRKY coding sequences, other WRKY transcriptional
regulatory regions can be isolated by obtaining regions 5' to the regions
of homology.

[0194] Methods are readily available in the art for the hybridization of
nucleic acid sequences. Promoter sequences from other plants may be
isolated according to well-known techniques based on their sequence
homology to the promoter sequences set forth herein. In these techniques,
all or part of the known transcriptional regulatory region sequence is
used as a probe, which selectively hybridizes to other sequences present
in a population of cloned genomic DNA, fragments (i.e.genomic libraries)
from a chosen organism.

[0195] For example, the entire transcriptional regulatory region or
portions thereof may be used as probes capable of specifically
hybridizing to corresponding promoter sequences. To achieve specific
hybridization under a variety of conditions, such probes include
sequences that are unique and are preferably at least about 10
nucleotides in length, and most preferably at least about 20 nucleotides
in length. Such probes may be used to amplify corresponding promoter
sequences from a chosen organism by the well-known process of polymerase
chain reaction (PCR). This technique may be used to isolate additional
promoter sequences from a desired organism or as a diagnostic assay to
determine the presence of the promoter sequence in an organism. Such
techniques include hybridization screening of plated DNA libraries
(either plaques or colonies; see e.g. Innis et al. (1990) PCR Protocols.
A Guide to Methods and Applications, eds., Academic Press).

[0196] The isolated transcriptional regulatory region of the present
invention can be modified to provide for a range of expression levels of
the heterologous nucleotide sequence. Thus, less than the entire region
may be utilized and the ability to drive pathogen or chemical-inducible
expression retained. However, it is recognized that expression levels of
mRNA may be altered and usually decreased with deletions of portions of
the region. Generally, at least about 20 nucleotides of an isolated
region will be used to drive expression of a nucleotide sequence.

[0197] It is recognized that to increase transcription levels enhancers
may be utilized in combination with the promoter regions of the
invention. Enhancers are nucleotide sequences that act to increase the
expression of a promoter region. Enhancers are known in the art. For
example, the enhancer from the cauliflower mosaic virus (CaMV) 35S
promoter has been isolated.

[0198] Modifications of the isolated transcriptional regulatory region of
the present invention can provide for a range of expression of the
heterologous nucleotide sequence. Thus, they may be modified to be weak
promoters or strong promoters. Generally, by "weak promoter" is intended
a promoter that drives expression of a coding sequence at a low level. By
"low level" is intended at levels of about {fraction (1/10,000)}
transcripts to about {fraction (1/100,000)} transcripts to about
{fraction (1/500,000)} transcripts. Conversely, a strong promoter drives
expression of a coding sequence at a high level, or at about {fraction
(1/10)} transcripts to about {fraction (1/100)} transcripts to about
{fraction (1/1000)} transcripts.

[0199] The nucleotide sequences for the transcriptional regulatory region
of the present invention may be the naturally occurring sequences or
sequences having substantial homology. By "substantial homology" is
intended a sequence exhibiting substantial functional and structural
equivalence with the naturally occurring sequence. Any structural
differences between substantially homologous sequences do not affect the
ability of the sequence to function as a promoter as disclosed in the
present invention. Thus, sequences having substantial sequence homology
with the sequence of the transcriptional regulatory region of the present
invention will direct expression during pathogen infection or chemical
induction of an operably linked heterologous nucleotide sequence. Two
transcriptional regulatory nucleotide sequences are considered
substantially homologous when they have at least about 70%, preferably at
least about 80%, more preferably at least about 90%, still more
preferably at least about 95% sequence homology. Substantially homologous
sequences of the present invention include variants of the disclosed
sequences such as those that result from site-directed mutagenesis, as
well as synthetically derived sequences.

[0200] Substantially homologous sequences of the present invention also
refer to those fragments of a particular promoter nucleotide sequence
disclosed herein that operate to promote the pathogen or
chemical-inducible expression of an operably linked heterologous
nucleotide sequence. These fragments will comprise at least about 20
contiguous nucleotides, or preferably 25, 30, 35, 40, 45, 50, 55, 60, 65,
70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or
1000 nucleotides of the transcriptional regulatory region of the present
invention. Such fragments may be obtained by use of restriction enzymes
to cleave the naturally occurring promoter nucleotide sequences disclosed
herein; by synthesizing a nucleotide sequence from the naturally
occurring promoter DNA sequence; or may be obtained through the use of
PCR technology. See particularly, Mullis et al. (1987) Methods Enzymol
155: 335-350, and Erlich, ed. (1989) PCR Technology (Stockton Press, New
York). Again, variants of these transcriptional regulatory region
fragments, such as those resulting from site-directed mutagenesis, are
encompassed by the compositions of the present invention.

[0201] Nucleotide sequences comprising at least about 20 contiguous
nucleotides of the sequence set forth in SEQ ID NO: 35 are encompassed.
These sequences may be isolated by hybridization, PCR, and the like. Such
sequences encompass fragments capable of driving developmentally
regulated expression, fragments useful as probes to identify similar
sequences, as well as elements responsible for temporal or tissue
specificity. Biologically active variants of the promoter sequences are
also encompassed by the method of the present invention. Such variants
should retain promoter activity, particularly the ability to drive
expression during flowering. Biologically active variants include, for
example, the native promoter sequences of the invention having one or
more nucleotide substitutions, deletions or insertions. Promoter activity
may be measured by Northern blot analysis, reporter activity measurements
when using transcriptional fusions, and the like. See, for example,
Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2.sup.nd
ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), herein
incorporated by reference.

[0202] The coding sequence expressed by the transcriptional regulatory
region of the invention may be used for expressing proteins during
pathogen infection or upon chemical induction with compounds such as
oxalic acid or salicylic acid. The affect of various expressed proteins
of interest include but are not limited to resistance to insects,
resistance to disease, resistance to stress, agronomic traits and the
like.

[0203] These results can be achieved by providing expression of
heterologous or increased expression of endogenous products in the plant.
Alternatively, the results can be achieved by providing for a reduction
of expression of one or more endogenous products, particularly enzymes
and cofactors in the plant. These changes result in a change in phenotype
of the transformed plant. For example, the transcriptional regulatory
regions of the invention can be used to express degradative enzymes that
are degrade toxins used by pathogens for invasion of a plant.
Alternatively, the transcriptional regulatory sequences of the invention
can be used to produce antisense mRNA complementary to the coding
sequence of an essential protein, inhibit production of a native protein
that is required or promotes pathogen invasion.

[0204] General categories of genes of interest for the purposes of the
present invention include for example, those genes involved in
information, such as Zinc fingers, those involved in communication, such
as kinases, and those involved in housekeeping, such as heat shock
proteins. It is recognized that the genes of interest depend on the exact
specificity of the WRKY transcriptional regulatory region.

[0205] More specific categories of transgenes, for example, include genes
involved in flowering; genes involved in resistance to disease,
pesticides and insect pests. It is recognized that any gene of interest
can be operably linked to the promoter of the inventions and expressed
during pathogen infection or upon chemical induction.

[0208] Exogenous products include plant enzymes and products as well as
those from other sources including prokaryotes and other eukaryotes. Such
products include enzymes, cofactors, hormones, and the like.

[0209] The heterologous nucleotide sequence operably linked to one of the
promoters disclosed herein may be an antisense sequence for a targeted
gene. By "antisense DNA nucleotide sequence" is intended a sequence that
is in inverse orientation to the 5'-to-3' normal orientation of that
nucleotide sequence. When delivered into a plant cell, expression of the
antisense DNA sequence prevents normal expression of the DNA nucleotide
sequence for the targeted gene. The antisense nucleotide sequence encodes
an RNA transcript that is complementary to and capable of hybridizing to
the endogenous messenger RNA (mRNA) produced by transcription of the DNA
nucleotide sequence for the targeted gene. In this case, production of
the native protein encoded by the targeted gene is invited to achieve a
desired phenotypic response. Thus, the promoter sequences disclosed
herein may be operably linked to antisense DNA sequence to reduce or
inhibit expression of a native protein in the plant.

[0210] Modulating polypeptide Levels and/or Composition

[0211] The present invention further provides a method for modulating
(i.e., increasing or decreasing) the concentration or composition of the
polypeptides of the present invention in a plant or part thereof.
Increasing or decreasing the concentration and/or the composition (i.e.,
the ratio of the polypeptides of the present invention) in a plant can
effect modulation. The method comprised introducing into a plant cell
with a recombinant expression cassette comprising a polynucleotide of the
present invention as described above to obtain a transformed plant cell,
culturing the transformed plant cell under plant cell growing conditions,
and inducing or repressing expression of a polynucleotide of the present
invention in the plant for a time sufficient to modulate concentration
and/or composition in the plant or plant part.

[0212] In some embodiments, the content and/or composition of polypeptides
of the present invention in a plant may be modulated by altering, in vivo
or in vitro, the promoter of a gene to up- or down-regulate gene
expression. In some embodiments, the coding regions of native genes of
the present invention can be altered via substitution, addition,
insertion, or deletion to decrease activity of the encoded enzyme. See,
e.g., Kmiec, U.S. Pat. No. 5,565,350; Zarling et al., PCT/US93/03868. And
in some embodiments, an isolated nucleic acid (e.g., a vector) comprising
a promoter sequence is transfected into a plant cell. Subsequently, a
plant cell comprising the promoter operably linked to a polynucleotide of
the present invention is selected for by means known to those of skill in
the art such as, but not limited to, Southern blot, DNA sequencing, or
PCR analysis using primers specific to the promoter and to the gene and
detecting amplicons produced therefrom. A plant or plant part altered or
modified by the foregoing embodiments is grown under plant forming
conditions for a time sufficient to modulate the concentration and/or
composition of polypeptides of the present invention in the plant. Plant
forming conditions are well known in the art and discussed briefly,
supra.

[0213] In general, concentration or composition is increased or decreased
by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% relative
to a native control plant, plant part, or cell lacking the aforementioned
recombinant expression cassette. Modulation in the present invention may
occur during and/or subsequent to growth of the plant to the desired
stage of development. Modulating nucleic acid expression temporally
and/or in particular tissues can be controlled by employing the
appropriate promoter operably linked to a polynucleotide of the present
invention in, for example, sense or antisense orientation as discussed in
greater detail, supra Induction of expression of a polynucleotide of the
present invention can also be controlled by exogenous administration of
an effective amount of inducing compound. Inducible promoters and
inducing compounds, which activate expression from these promoters, are
well known in the art. In preferred embodiments, the polypeptides of the
present invention are modulated in monocots, particularly maize.

[0214] Molecular Markers

[0215] The present invention provides a method of genotyping a plant
comprising a polynucleotide of the present invention. Optionally, the
plant is a monocot, such as maize or sorghum. Genotyping provides a means
of distinguishing homologs of a chromosome pair and can be used to
differentiate segregants in a plant population. Molecular marker methods
can be used for phylogenetic studies, characterizing genetic
relationships among crop varieties, identifying crosses or somatic
hybrids, localizing chromosomal segments affecting monogenic traits, map
based cloning, and the study of quantitative inheritance. See, e.g.,
Plant Molecular Biology: A Laboratory Manual, Chapter 7, Clark, Ed.,
Springer-Verlag, Berlin (1997). For molecular marker methods, see
generally, The DNA Revolution by Andrew H. Paterson 1996 (Chapter 2) in:
Genome Mapping in plants (ed. Andrew H. Paterson) by Academic Press/R. G.
Lands Company, Austin, Tex., pp. 7-21.

[0216] The particular method of genotyping in the present invention may
employ any number of molecular marker analytic techniques such as, but
not limited to, restriction fragment length polymorphism's (RFLPs). RFLPs
are the product of allelic differences between DNA restriction fragments
resulting from nucleotide sequence variability. As is well known to those
of skill in the art, RFLPs are typically detected by extraction of
genomic DNA and digestion with a restriction enzyme. Generally, the
resulting fragments are separated according to size and hybridized with a
probe; single copy probes are preferred. Restriction fragments from
homologous chromosomes are revealed. Differences in fragment size among
alleles represent an RFLP. Thus, the present invention further provides a
means to follow segregation of a gene or nucleic acid of the present
invention as well as chromosomal sequences genetically linked to these
genes or nucleic acids using such techniques as RFLP analysis. Linked
chromosomal sequences are within 50 centiMorgans (cM), often within 40 or
30 cM, preferably within 20 or 10 cM, more preferably within 5, 3, 2, or
1 cM of a gene of the present invention.

[0217] In the present invention, the nucleic acid probes employed for
molecular marker mapping of plant nuclear genomes selectively hybridize,
under selective hybridization conditions, to a gene encoding a
polynucleotide of the present invention. In preferred embodiments, the
probes are selected from polynucleotides of the present invention.
Typically, these probes are cDNA probes or restriction enzyme treated
(e.g., PST 1) genomic clones. The length of the probes is discussed in
greater detail, supra, but is typically at least 15 bases in length, more
preferably at least 20, 25, 30, 35, 40, or 50 bases in length. Generally,
however, the probes are less than about 1 kilobase in length. Preferably,
the probes are single copy probes that hybridize to a unique locus in
haploid chromosome compliment. Some exemplary restriction enzymes
employed in RFLP mapping are EcoRI, EcoRv, and SstI. As used herein the
term "restriction enzyme" includes reference to a composition that
recognizes and, alone or in conjunction with another composition, cleaves
at a specific nucleotide sequence.

[0218] The method of detecting an RFLP comprises the steps of (a)
digesting genomic DNA of a plant with a restriction enzyme; (b)
hybridizing a nucleic acid probe, under selective hybridization
conditions, to a sequence of a polynucleotide of the present of said
genomic DNA; (c) detecting therefrom a RFLP. Other methods of
differentiating polymorphic (allelic) variants of polynucleotides of the
present invention can be had by utilizing molecular marker techniques
well known to those of skill in the art including such techniques as: 1)
single stranded conformation analysis (SSCA); 2) denaturing gradient gel
electrophoresis (DGGE); 3) RNase protection assays; 4) allele-specific
oligonucleotides (ASOs); 5) the use of proteins which recognize
nucleotide mismatches, such as the E. coli mutS protein; and 6)
allele-specific PCR. Other approaches based on the detection of
mismatches between the two complementary DNA strands include clamped
denaturing gel electrophoresis (CDGE); heteroduplex analysis (HA); and
chemical mismatch cleavage (CMC). Thus, the present invention further
provides a method of genotyping comprising the steps of contacting, under
stringent hybridization conditions, a sample suspected of comprising a
polynucleotide of the present invention with a nucleic acid probe.
Generally, the sample is a plant sample, preferably, a sample suspected
of comprising a maize polynucleotide of the present invention (e.g.,
gene, mRNA). The nucleic acid probe selectively hybridizes, under
stringent conditions, to a subsequence of a polynucleotide of the present
invention comprising a polymorphic marker. Selective hybridization of the
nucleic acid probe to the polymorphic marker nucleic acid sequence yields
a hybridization complex. Detection of the hybridization complex indicates
the presence of that polymorphic marker in the sample. In preferred
embodiments, the nucleic acid probe comprises a polynucleotide of the
present invention.

[0221] Further, the polypeptide-encoding segments of the polynucleotides
of the present invention can be modified to alter codon usage. Altered
codon usage can be employed to alter translational efficiency and/or to
optimize the coding sequence for expression in a desired host such as to
optimize the codon usage in a heterologous sequence for expression in
maize. Codon usage in the coding regions of the polynucleotides of the
present invention can be analyzed statistically using commercially
available software packages such as "Codon Preference" available form the
University of Wisconsin Genetics Computer Group (see Devereaux et al.,
Nucleic Acids Res. 12:387-395 (1984)) or MacVector 4.1 (Eastman Kodak
Co., New Haven, Conn.). Thus, the present invention provides a codon
usage frequency characteristic of the coding region of at least one of
the polynucleotides of the present invention. The number of
polynucleotides that can be used to determine a codon usage frequency can
be any integer from 1 to the number of polynucleotides of the present
invention as provided herein. Optionally, the polynucleotides will be
full-length sequences. An exemplary number of sequences for statistical
analysis can be at least 1, 5, 10, 20, 50, or 100.

[0222] Sequence Shuffling

[0223] The present invention provides methods for sequence shuffling using
polynucleotides of the present invention, and compositions resulting
therefrom. Sequence shuffling is described in PCT publication No. WO
96/19256. See also, Zhang, J. -H., et al. Proc. Natl. Acad. Sci. USA
94:4504-4509 (1997). Generally, sequence shuffling provides a means for
generating libraries of polynucleotides having a desired characteristic,
which can be selected or screened for. Libraries of recombinant
polynucleotides are generated from a population of related sequence
polynucleotides, which comprise sequence regions, which have substantial
identity and can be homologously recombined in vitro or in vivo. The
population of sequence-recombined polynucleotides comprises a
subpopulation of polynucleotides which possess desired or advantageous
characteristics and which can be selected by a suitable selection or
screening method. The characteristics can be any property or attribute
capable of being selected for or detected in a screening system, and may
include properties of: an encoded protein, a transcriptional element, a
sequence controlling transcription, RNA processing, RNA stability,
chromatin conformation, translation, or other expression property of a
gene or transgene, a replicative element, a protein-binding element, or
the like, such as any feature which confers a selectable or detectable
property. In some embodiments, the selected characteristic will be a
decreased K.sub.m and/or increased K.sub.cat over the wild-type protein
as provided herein. In other embodiments, a protein or polynucleotide
generated from sequence shuffling will have a ligand binding affinity
greater than the non-shuffled wild-type polynucleotide. The increase in
such properties can be at least 110%, 120%, 130%, 140%, or at least 150%
of the wild-type value.

[0224] Generic and Consensus Sequences

[0225] Polynucleotides and polypeptides of the present invention further
include those having: (a) a generic sequence of at least two homologous
polynucleotides or polypeptides, respectively, of the present invention;
and, (b) a consensus sequence of at least three homologous
polynucleotides or polypeptides, respectively, of the present invention.
The generic sequence of the present invention comprises each species of
polypeptide or polynucleotide embraced by the generic polypeptide or
polynucleotide, sequence, respectively. The individual species
encompassed by a polynucleotide having an amino acid or nucleic acid
consensus sequence can be used to generate antibodies or produce nucleic
acid probes or primers to screen for homologs in other species, genera,
families, orders, classes, phylums, or kingdoms. For example, a
polynucleotide having a consensus sequence from a gene family of Zea mays
can be used to generate antibody or nucleic acid probes or primers to
other Gramineae species such as wheat, rice, or sorghum. Alternatively, a
polynucleotide having a consensus sequence generated from orthologous
genes can be used to identify or isolate orthologs of other taxa.
Typically, a polynucleotide having a consensus sequence will be at least
9, 10, 15, 20, 25, 30, or 40 amino acids in length, or 20, 30, 40, 50,
100, or 150 nucleotides in length. As those of skill in the art are
aware, a conservative amino acid substitution can be used for amino
acids, which differ amongst aligned sequence but are from the same
conservative amino substitution group as discussed above. Optionally, no
more than 1 or 2 conservative amino acids are substituted for each 10
amino acid length of consensus sequence.

[0226] Similar sequences used for generation of a consensus or generic
sequence include any number and combination of allelic variants of the
same gene, orthologous, or paralogous sequences as provided herein.
Optionally, similar sequences used in generating a consensus or generic
sequence are identified using the BLAST algorithm's smallest sum
probability (P(N)). Various suppliers of sequence-analysis software are
listed in chapter 7 of Current Protocols in Molecular Biology, F. M.
Ausubel et al., Eds. Current Protocols, a joint venture between Greene
Publishing Associates, Inc. and John Wiley & Sons, Inc. (Supplement 30).
A polynucleotide sequence is considered similar to a reference sequence
if the smallest sum probability in a comparison of the test nucleic acid
to the reference nucleic acid is less then about 0.1, more preferably
less than about 0.01, or 0.001, and most preferably less than about
0.0001, or 0.00001. Similar polynucleotides can be aligned and a
consensus or generic sequence generated using multiple sequence alignment
software available from a number of commercial suppliers such as the
Genetics Computer Group's (Madison, Wis.) PILEUP software, Vector NTI's
(North Bethesda, Md.) ALIGNX, or Genecode's (Ann Arbor, Mich.) SEQUENCER.
Conveniently, default parameters of such software can be used to generate
consensus or generic sequences.

[0227] Use of Subsequences of WRKY Polynucleotides

[0228] As previously discussed, WRKY polynucleotides have conserved
domains. The binding specificity of the WRKY domains is a hallmark of a
specific set of promoters that a particular WRKY interacts with.
Therefore, a subsequence of a WRKY polynucleotide could be utilized in
the following manner.

[0229] First, a subsequence of WRKY could be expressed in an expression
system (please see the section entitled "Expression of Proteins in Host
Cells"), such as an E. coli expression system. The ability of the
expressed protein could then be tested for its ability to bind target DNA
in a gel shift experiment or other interaction assay. Either specific
candidate promoter DNA or total genomic DNA could be used in the
experiment.

[0230] Alternatively, a subsequence of a WRKY polynucleotide could be
fused in frame to an N-terminal DNA activation domain, such as, but not
limited to, a myb or myc homolog or the activation domain of another
WRKY. The fusion polynucleotide would then be expressed in an expression
system, such as, but not limited to, a transient or stable plant
expression system. Specific promoters could then be identified or global
transcript profiling could be used to identify genes and their associated
promoters that respond to the WRKY domain/activation domain fusion.

[0231] Although the present invention has been described in some detail by
way of illustration and example for purposes of clarity of understanding,
it will be obvious that certain changes and modifications may be
practices within the scope of the appended claims.

EXAMPLE 1

Isolation of Maize ZmWRKY3-1 cDNA

[0232] Using the techniques described above a partial sequence of a
homolog of parsley WRKY3 was found in a maize cDNA library. A cDNA
library was made from mRNA isolated from maize cells. The maize cells
were treated with water or 1.times.10.sup.6 spores/ml of Fusarium
moniliforme. Cells were harvested 2 and 6 hours after treatment. Total
RNA was isolated using Tri-Reagent.TM. and mRNA was isolated using
PolyAtract.TM. (Promega). Zap-cDNA synthesis kit (Stratagene) was used to
prepare cDNA, which was cloned into HybriZap.RTM. (Stratagene). The
primary library was amplified and phagemid was excised from the secondary
library. The phagemid prep was amplified in XLOLR cells and purified
(Qiagen). All library manipulations were performed according to the
HybriZap.RTM. manual.

[0234] Gene identities can be determined by conducting BLAST (Basic Local
Alignment Search Tool; Altschul, S. F., et al., (1993) J. Mol. Biol.
215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/) searches under default
parameters for similarity to sequences contained in the BLAST "nr"
database (comprising all non-redundant GenBank CDS translations,
sequences derived from the 3-dimensional structure Brookhaven Protein
Data Bank, the last major release of the SWISS-PROT protein sequence
database, EMBL, and DDBJ databases). The cDNA sequences are analyzed for
similarity to all publicly available DNA sequences contained in the "nr"
database using the BLASTN algorithm. The DNA sequences are translated in
all reading frames and compared for similarity to all publicly available
protein sequences contained in the "nr" database using the BLASTX
algorithm (Gish, W. and States, D. J. Nature Genetics 3:266-272 (1993))
provided by the NCBI. In some cases, the sequencing data from two or more
clones containing overlapping segments of DNA are used to construct
contiguous DNA sequences.

[0235] Additional maize WRKY sequences were identified from a cDNA library
generated and sequenced as described below. Total RNA was isolated from
corn tissues with TRIzol Reagent (Life Technology Inc. Gaithersburg, Md.)
using a modification of the guanidine isothiocyanate/acid-phenol
procedure described by Chomczynski and Sacchi (Chomczynski, P., and
Sacchi, N. Anal. Biochem. 162, 156 (1987)). In brief, plant tissue
samples were pulverized in liquid nitrogen before the addition of the
TRIzol Reagent, and then were further homogenized with a mortar and
pestle. Addition of chloroform followed by centrifugation was conducted
for separation of an aqueous phase and an organic phase. The total RNA
was recovered by precipitation with isopropyl alcohol from the aqueous
phase.

[0236] The selection of poly(A)+ RNA from total RNA was performed using
PolyATact system (Promega Corporation, Madison Wis.). In brief,
biotinylated oligo(dT) primers were used to hybridize to the 3' poly(A)
tails on mRNA. The hybrids were captured using streptavidin coupled to
paramagnetic particles and a magnetic separation stand. The mRNA was
washed at high stringent condition and eluted by RNase-free deionized
water.

[0237] cDNA synthesis was performed and unidirectional cDNA libraries were
constructed using the SuperScript Plasmid System (Life Technology Inc.
Gaithersburg, Md.). The first strand of cDNA was synthesized by priming
an oligo(dT) primer containing a Not I site. The reaction was catalyzed
by SuperScript reverse Transcriptase II at 45.degree. C. The second
strand of cDNA was labeled with alpha-.sup.32P-dCTP and a portion of the
reaction was analyzed by agarose gel electrophoresis to determine cDNA
sizes. cDNA molecules smaller than 500 base pairs and unligated adaptors
were removed by Sephacryl-S400 chromatography. The selected cDNA
molecules were ligated into a pSPORT1 vector between the NotI and SalI
sites.

[0238] Individual colonies were picked and DNA was prepared either by PCR
with Ml 3 forward primers and M13 reverse primers, or by plasmid
isolation. All the cDNA clones were sequenced using M13 reverse primers.

[0239] cDNA libraries subjected to the subtraction procedure were plated
out on 22.times.22 cm.sup.2 agar plate at density of about 3,000 colonies
per plate. The plates were incubated in a 37.degree. C. incubator for
12-24 hours. Colonies were picked into 384-well plates by a robot colony
picker, Q-bot (GENETIX Limited). These plates were incubated overnight at
37.degree. C.

[0240] Once sufficient colonies were picked, they were pinned onto
22.times.22 cm.sup.2 nylon membranes using Q-bot. Each membrane contained
9,216 colonies or 36,864 colonies. These membranes were placed onto agar
plate with appropriate antibiotic. The plates were incubated at
37.degree. C. for overnight.

[0241] After colonies were recovered on the second day, these filters were
placed on filter paper prewetted with denaturing solution for four
minutes, then were incubated on top of a boiling water bath for
additional four minutes. The filters were then placed on filter paper
prewetted with neutralizing solution for four minutes. After excess
solution was removed by placing the filters on dry filter papers for one
minute, the colony site of the filters were placed into Proteinase K
solution, incubated at 37.degree. C. for 40-50 minutes. The filters were
placed on dry filter papers to dry overnight. DNA was then cross-linked
to nylon membrane by UV light treatment.

[0242] Colony hybridization was conducted as described by Sambrook, J.,
Fritsch, E. F. and Maniatis, T., (in Molecular Cloning: A laboratory
Manual, 2.sup.nd Edition). The following probes were used in colony
hybridization:

[0243] 1. First strand cDNA from the same tissue as the library was made
from to remove the most redundant clones.

[0244] 2. 48-192 most redundant cDNA clones from the same library based on
previous sequencing data.

[0248] The image of the autoradiography was scanned into computer and the
signal intensity and cold colony addresses of each colony was analyzed,
re-arraying of cold-colonies from 384 well plates to 96 well plates was
conducted using Q-bot. The cDNA sequence information generated from the
cDNA library was then analyzed by BLAST to find additional maize WRKY
polynucleotides.

[0249] The following maize WRKY polynucleotides were found as described
above. ZmWRKY1-1 polynucleotide is shown in SEQ ID NO: 37. The protein
translation of ZmWRKY1-1 is shown in SEQ ID NO: 38. The ZmWRKY1-2
polynucleotide is shown in SEQ ID NO: 39. The ZmWRKY2-2 polynucleotide is
shown in SEQ ID NO: 40. The ZmWRKY3-3 polynucleotide is shown in SEQ ID
NO: 41. The ZmWRKY3-4 polynucleotide is shown in SEQ ID NO: 42. The
ZmWRKY3-5 polynucleotide is shown in SEQ ID NO: 43.

[0250] Northern Blot Assay

[0251] The mRNA steady-state level of maize WRKY1 and WRKY3 were studied
after treatment with Fusarium moniliforme spores. Mid-log maize GS3
suspension cell cultures (75 ml) were treated with 1 ml of Fusarium
spores to give a concentration of 1,000,000 spores/ml. Control cultures
were treated with 1 ml of water. The cultures were harvested at 0, 1, and
3 hours post-treatment. RNA was extracted and Northern Blot analysis was
performed according to Church, et al., Proc. Natl. Acad. Sci. USA
81:1991-1995 (1984). The blots were probed with DNA that was either
ZmWRKY1-(SEQ ID NO: 37) or ZmWRKY3-1 (SEQ ID NO: 1). At 1 and 3 hours
post-treatment there was a significant induction of both ZmWRKY1-1 and
ZmWRKY3-1, substantiating the role of ZmWRKY1-1 and ZmWRKY3-1 in a plants
response to pathogen infection.

[0252] Transgenic Evaluation of ZmWRKY3-1

[0253] The promoter region of ZmPR-1 gene (PCT Publication WO 99/43819)
was fused with the coding sequence of a .beta.-glucuronidase (GUS)
reporter gene resulting in a molecular marker construct (ZmPR-1::GUS).
The coding sequences of ZmNPR1 (PCT Publication number WO 00/65037) and
ZmWRKY3-1 driven by the ubiquitin promoter were employed as regulator
constructs (Ubi::ZmNPR1 and Ubi::ZmWRKY3). Act::luciferase (rice actin
promoter (U.S. Pat. No. 5,641,876) operably linked to the luciferase gene
from the Promega Dual-luciferase reporter assay system) was used as an
internal standard for normalization of the variation inherent in
bombardment. A DNA carrier construct was also included to maintain
uniform DNA concentrations.

[0254] Maize immature embryos (IE) were co-bombarded with the marker
construct and either the DNA carrier construct or the regulator
construct. The internal standard was also included in all bombardments.
Mixture of DNA from 20 .mu.l of ZmPR-1::GUS at 0.05 .mu.g/.mu.l, 5 .mu.l
of the regulator or carrier DNA (1.0 .mu.g/.mu.l), and 10 .mu.l of
Act::luciferase at 0.1 .mu.g/.mu.l were co-precipitated with 70 .mu.l of
2.5 M CaC1.sub.2 and 20 .mu.l of 0.1 M spermidine onto 50 .mu.l of
tungsten particles (1.0 .mu.m at a particle density of 15 mg/ml). For
each bombardment, 45 IEs were placed on a high osmotic medium (12 g/L
sucrose) plate for 4 hours before the bombardment. After the bombardment
the IEs were placed in culture on the same osmotic medium for 24 hours
and then divided into three groups. One group was cultured on a piece of
filter paper wetted with the same osmotic medium without any addition of
signal molecules as a control and the other two were cultured under the
same condition but the medium contained either 1 mM SA or 0.1 mM JA. All
IEs were cultured for another 24 hours.

[0255] Three IEs from each group were histochemically stained in X-Gluc
staining solution for overnight at 37.degree. C. The rest of the IEs were
subjected to GUS fluorometric and luciferase assays. Fluorometric
measurements of GUS activity were performed by using 50 .mu.l protein
extract prepared from the 12 IEs of each treatment and quantified in
Fluoroskan Ascent FL (Labsystem) for two time points, 10 and 30 min.
Luciferase activity was quantified in a Monolight 2010 (Analytical
Luminescence Lab) by mixing 20 .mu.l of protein extract with 100 .mu.l of
reaction buffer (Dual-Luciferase Reporter Assay System, Promega) and
taking the measurements after 10 seconds. To normalize promoter/marker
activity, the GUS value detected in each sample was divided by the
luciferase value obtained in the same bombarded sample treated without
signal molecules.

[0256] It has been established in Arabidopsis that SA and NPR1 are two key
regulators that activate the SA-dependent SAR response. Both
histochemical and fluorometric GUS assay results showed that ZmPR-1::GUS
expression was induced by more than 3-fold by SA treatment alone, as well
as in cells over-expressing ZmNPR1 alone.

[0257] In contrast, cells expressing WRKY3-1 showed complete suppression
of GUS activity under both JA treatment and no treatment. An antagonistic
relationship between the SA- and JA-dependent plant defense signaling
transduction pathways has been shown in several reports. WRKY factors
have been proposed as repressors of PR-1 expression. The results indicate
that JA and ZmWRKY3-1 suppress ZmPR-1::GUS expression in maize. Thus,
ZmWRKY3-1 functions in suppression of ZmPR-1 in a transient system. This
suppression of ZmPR-1 is consistent with what is expected for at least
certain WRKY genes and is a further indicator of the role ZmWRKY3-1 plays
in a plant's defense to disease.

[0258] Therefore, to modulate the level of disease resistance in a plant
using a WRKY polynucleotide, it may be necessary to inhibit or lower the
expression of the native WRKY gene or in the alternative increase
expression by overexpression of the transgene, depending the disease
resistance pathway to be modified. Methods of decreasing expression of a
gene in a plant are well known in the art. For example, reduction in the
expression of a WRKY gene can be accomplished by a number of methods,
including but not limited to, antisense, catalytic RNA molecules
(ribozymes), cross-linking agents, alkylating agents, radical generating
species, or sense suppression. A discussion of these methods can be found
in the section entitled "Recombinant Expression Cassettes." If
suppression of WRKY is only desired during pathogen infection, then a
pathogen inducible promoter operably linked to the WRKY polynucleotide in
the sense orientation for sense suppression or antisense orientation for
antisense suppression may be used. Alternatively a constitutive promoter
operably linked to a WRKY polynucleotide in the sense or antisense
orientation may be used. The recombinant expression cassette can then be
transformed into plant cells and a whole plant can be regenerated.

[0259] Alternatively, the native WRKY gene can be modified by chimeric
oligonucleotides. U.S. Pat. No. 5,565,350 describes chimeric
oligonucleotides that are useful for targeted gene correction and methods
for their use in cultured mammalian cells. The use of chimeric
oligonucleotides in plants is described in PCT Publication No. WO
99/25853, published May 27, 1999. Both disclosures are herein
incorporated by reference.

[0260] In addition, the expression of WRKY gene may be reduced by the use
of hairpin dsRNA techniques. These techniques are illustrated in PCT
published applicant No. WO 99/53050, published Oct. 21, 1999 and WO
98/53083 published Nov. 26, 1998, both of which are herein incorporated
by reference.

EXAMPLE 2

Isolation of Sunflower WRKY Polynucleotides (SWRKY1)

[0261] Fungal Infection and Chemical Treatments:

[0262] Sunflower plants (SMF3) were planted in 4-inch pot and grown in
greenhouse for first four weeks. After transfer to growth chamber, plants
were maintained under a 12-hour photoperiod at 22.degree. C. with an 80%
relative humidity. Six-week old plants were inoculated with
Sclerotinia-infected carrot plugs or sprayed with four different
chemicals at the given concentration. For each plant, three petioles were
inoculated and wrapped with lx2 inch parafilm. Plant tissue samples were
harvested at different time points and immediately frozen in liquid
nitrogen and then stored at -80.degree. C.

[0263] Construction of the Sclerotinia-infected and Resistance-enhanced
Sunflower cDNA Libraries:

[0264] Six-week old SMF3 sunflower plants were infected with Sclerotinia
sclerotrium by petiole inoculation with Sclerotinia-infested carrot
plugs. Six days after infection, leaf and stem tissues were collected
from infected plants for total RNA isolation. Total RNA was also isolated
from transgenic sunflower plants expressing a wheat oxalate oxidase gene
at the 6-week stage (U.S. Pat. No. 6,166,291; and hereby incorporated by
reference). Previous studies have showed that elevated levels of
H.sub.2O.sub.2, SA and PR1 protein were detected in oxalate oxidase
expressing transgenic plants at the 6-week stage and that the plants
showed more resistant to Sclerotinia infection (U.S. Pat. No. 6,166,291).
The mRNAs were isolated by a mRNA purification kit (BRL) according to
manufacture's instruction. The cDNA libraries were constructed with the
ZAP-cDNA synthesis kit into pBluescript phagemid (Stratagene). A cDNA
library mixture for PCR cloning was made of oxalate oxidase transgenic
stem and Sclerotinia-infected leaf libraries (1:2 mix).

[0265] PCR amplification of Sunflower WRKY Genes:

[0266] To isolate sunflower WRKY genes, a conserved motif (WRKYGQK) of
zinc-finger type transcriptional factor was used to design four
degenerate primers:

[0267] W-s1: 5'-TGGMGNAARTAYGGNCAGAA-3' (SEQ ID NO: 3)

[0268] W-s2: 5'-TGGMGNAARTAYGGNCAAAA-3' (SEQ ID NO: 4)

[0269] W-as1: 5'-TTYTGNCCRTAYTTNCGCCA-3' (SEQ ID NO: 5)

[0270] W-as2: 5'-TTYTGNCCRTAYTTNCTCCA-3' (SEQ ID NO: 6)

[0271] Primers for Library Vector (pBS)

[0272] PBS-upper: GCGATTAAGTTGGGTAACGCCAGGGT (SEQ ID NO: 7)

[0273] PBS-lower: TCCGGCTCGTATGTTGTGTGGAATTG (SEQ ID NO: 8)

[0274] The cDNA library was used as the DNA template for PCR
amplification. To facilitate the cloning process, a pair of 28 base pair
vector primers of flanking cDNA (3' and 5') of pBS vector were designed.
The primers were directionally amplified with either the 5' or 3' end of
the cDNA of the vector primers (pBS-upper or pBS-lower) paired with a
degenerate primer. The full-length cDNA was amplified using a new gene
specific primer containing the region upstream of the ATG start sequence
and the vector primer at the 3' end.

[0277] Amplified PCR fragments with the expected sizes were individually
sliced out of the gel for a second round of PCR re-amplification with the
same condition as initial PCR. Each second round of PCR product showing a
single band with the expected size was cloned into a TA vector (Clontech)
according to the supplier's instructions. Positive clones were sequenced
using an Applied Biosystems 373A automated sequencer. DNA sequence
analysis was carried out with Sequencer (3.0). Multiple-sequence
alignments of the DNA sequence were carried out using CLUSTAL W
(Thompson, et al., Nuc. Acids Res. 22:4673-80 (1994)).

[0278] Results

[0279] Four sunflower WRKY homologs have been cloned and sequenced. The
SWRKY1-1 polynucleotide and polypeptide sequence is shown in SEQ ID NOS:
9 and 10. SWRKY1-2 polynucleotide and polypeptide sequence is shown in
SEQ ID NOS: 11 and 12. SWRKY1-3 polynucleotide and polypeptide sequence
is shown in SEQ ID NOS: 13 and 14. SWRKY1-4 polynucleotide and
polypeptide sequence is shown in SEQ ID NOS: 15 and 16. BLAST search
results indicates that all four cDNAs were homologous to parsley WRKY1
gene. Amino acid sequence alignment and genetic distance analysis reveals
that three of the sunflower WRKY genes (SWRKY1-3, 1-2 and 1-4) are very
closely related. Sunflower WRKY1-1 is less similar to the other sunflower
WRKY genes but is closer in homology to the parsley WRKY1 gene.

[0280] Northern Blot Assay

[0281] The mRNA steady-state level of sunflower WRKY1 was studied under
different chemical treatments. Six-week-old sunflower plants were sprayed
with oxalic acid (OA) (5 mM), hydrogen peroxide (5 mM), salicylic acid
(SA) (5 mM) and jasmonic acid (JA) (45 uM in 0.1% ethanol). Leaf samples
were collected at 0, 6, 12, and 24 hours after application and
immediately frozen in liquid nitrogen. Twenty microgram of total RNA were
loaded in each sample lane. Control tissue was SMF3 leaf tissue with no
treatment. Northern Blot analysis was performed according to Church, et
al., Proc. Natl. Acad Sci. USA 81:1991-1995 (1984). The blots were probed
with DNA from the sunflower WRKY1-1 polynucleotide. The salicylic acid
and oxalic acid treatments showed significant induction of WRKY1-1 within
6 hours. The hydrogen peroxide and jasmonic acid treatments did not
induce WRKY1-1 RNA within 6 hours.

[0282] The mRNA steady-state level of sunflower WRKY1 gene was also
studied under Sclerotinia-infection and oxalate oxidase expression.
Six-week-old transgenic sunflower leaf and stem samples were collected
along with control SMF3 samples. Sclerotinia-infected samples were
harvested on 6 days after inoculation. Twenty microgram of total RNA were
loaded in each sample lane. Northern Blot analysis was performed
according to Church, et al., Proc. Natl. Acad Sci. USA 81:1991-1995
(1984). The blots were probed with sunflower WRKY1-1 polynucleotide.
Sunflower WRKY1-1 was induced by Sclerotinia infection and oxalate
oxidase expression in sunflower.

[0286] Amplified PCR fragments with the expected sizes were individually
sliced out of the gel for a second round PCR re-amplification with the
same condition as the initial PCR. Each second round PCR product showing
a single band with the expected size was cloned into TA vector
(Invitrogen) according to the supplier's instructions. Identified
positive clones were selected for DNA sequencing using an Applied
Biosystems 373A (ABI) automated sequencer. DNA sequence analysis was
carried out with Sequencer (3.0).

[0291] The BLASTX search using the sequences from clone r1s24.pk0005.d1
revealed similarity of the proteins encoded by the cDNAs to WRKY1 from
Petroselinum crispum (NCBI Accession No. 1431872) with a pLog score of
26.22. The sequence of a portion of the cDNA insert from clone
r1s24.pk0005.d1 is shown in SEQ ID NO: 17; the deduced amino acid
sequence of this cDNA is shown in SEQ ID NO: 18. BLAST scores and
probabilities indicate that the instant nucleic acid fragments encode
portions of WRKY1. These sequences represent the first rice sequence
encoding WRKY1.

[0292] The BLASTX search using the sequences from clone rdr1f.pk004.m4
revealed similarity of the proteins encoded by the cDNAs to WRKY3 from
Avena sativa (NCBI Accession No. 4894963) with a pLog score of 28.00. The
sequence of a portion of the cDNA insert from clone rdr1f.pk004.m4 is
shown in SEQ ID NO: 19; the deduced amino acid sequence of this cDNA is
shown in SEQ ID NO: 20. BLAST scores and probabilities indicate that the
instant nucleic acid fragments encode portions of WRKY3. These sequences
represent the first rice sequence encoding WRKY3.

[0294] The BLASTX search using the sequences from clone srr3c.pk001.a20
revealed similarity of the proteins encoded by the cDNAs to WRKY1 from
Nicotiana tabacum (NCBI Accession No. 5360683) with a pLog score of
28.40. The sequence of a portion of the cDNA insert from clone
srr3c.pk001.a20 is shown in SEQ ID NO: 21; the deduced amino acid
sequence of this cDNA is shown in SEQ ID NO: 22. BLAST scores and
probabilities indicate that the instant nucleic acid fragments encode
portions of WRKY1. These sequences represent the first soybean sequence
encoding WRKY1.

[0295] The BLASTX search using the sequences from clone sfll.pk0008.a2
revealed similarity of the proteins encoded by the cDNAs to WRKY2 from
Petroselinum crispum (NCBI Accession No. 1432058) with a pLog score of
70.70. The sequence of a portion of the cDNA insert from clone
sfll.pk0008.a2 is shown in SEQ ID NO: 23; the deduced amino acid sequence
of this cDNA is shown in SEQ ID NO: 24. BLAST scores and probabilities
indicate that the instant nucleic acid fragments encode portions of
WRKY2. These sequences represent the first soybean sequence encoding
WRKY2.

[0296] The BLASTX search using the sequences from clone sdp4c.pk007.b19
revealed similarity of the proteins encoded by the cDNAs to WRKY3 from
Nicotiana tabacum (NCBI Accession No. 4760596) with a pLog score of
28.10. The sequence of a portion of the cDNA insert from clone
sdp4c.pk007.b19 is shown in SEQ ID NO: 25; the deduced amino acid
sequence of this cDNA is shown in SEQ ID NO: 26. BLAST scores and
probabilities indicate that the instant nucleic acid fragments encode
portions of WRKY3. These sequences represent the first soybean sequence
encoding WRKY3.

[0297] Characterization of cDNA Clones Encoding Wheat WRKY2 and WRKY3

[0298] The BLASTX search using the sequences from clone wlk4.pk0012.c10
revealed similarity of the proteins encoded by the cDNAs to WRKY2 from
Nicotiana tabacum (NCBI Accession No. 4760692) with a pLog score of
87.70. The sequence of a portion of the cDNA insert from clone
wlk4.pk0012.c10 is shown in SEQ ID NO: 27; the deduced amino acid
sequence of this cDNA is shown in SEQ ID NO: 28. BLAST scores and
probabilities indicate that the instant nucleic acid fragments encode
portions of WRKY2. These sequences represent the first wheat sequence
encoding WRKY2.

[0299] The BLASTX search using the sequences from clone wlmk8.pk0019.b11
revealed similarity of the proteins encoded by the cDNAs to WRKY3 from
Avena sativa (NCBI Accession No. 4894963) with a pLog score of 148.00.
The sequence of a portion of the cDNA insert from clone wlmk8.pkOOl9.bl 1
is shown in SEQ ID NO: 29; the deduced amino acid sequence of this cDNA
is shown in SEQ ID NO: 30. BLAST scores and probabilities indicate that
the instant nucleic acid fragments encode portions of WRKY3. These
sequences represent the first wheat sequence encoding WRKY3.

[0301] The BLASTX search using the sequences from clone cr1n.pk0183.d7
revealed similarity of the proteins encoded by the cDNAs to WRKY2-1 from
Petroselinum crispum (NCBI Accession No. 1432058) with a pLog score of
47.22. The sequence of a portion of the cDNA insert from clone
cr1n.pk0183.d7 is shown in SEQ ID NO: 31; the deduced amino acid sequence
of this cDNA is shown in SEQ ID NO: 32. BLAST scores and probabilities
indicate that the instant nucleic acid fragments encode portions of
WRKY2-1. These sequences represent the first maize sequence encoding
WRKY2-1.

[0302] The BLASTX search using the sequences from clone cpk1c.pk001.f20
revealed similarity of the proteins encoded by the cDNAs to WRKY3 from
Nicotiana tabacum (NCBI Accession No. 4760596) with a pLog score of
15.70. The sequence of a portion of the cDNA insert from clone
cpk1c.pk001.f20 is shown in SEQ ID NO: 33; the deduced amino acid
sequence of this cDNA is shown in SEQ ID NO: 34. BLAST scores and
probabilities indicate that the instant nucleic acid fragments encode
portions of WRKY3-2. These sequences represent the first maize sequence
encoding WRKY3-2.

EXAMPLE 4

[0303] Transformation and Regeneration of Transgenic Maize Plants

[0304] Immature maize embryos from greenhouse donor plants are bombarded
with a plasmid containing a WRKY sequences of the present invention
operably linked to a ubiquitin promoter and the selectable marker gene
PAT (Wohlleben et al. (1988) Gene 70:25-37), which confers resistance to
the herbicide Bialophos. Alternatively, the selectable marker gene is
provided on a separate plasmid. Transformation is performed as follows.
Media recipes follow below.

[0305] Preparation of Target Tissue

[0306] The ears are husked and surface sterilized in 30% Clorox bleach
plus 0.5% Micro detergent for 20 minutes, and rinsed two times with
sterile water. The immature embryos are excised and placed embryo axis
side down (scutellum side up), 25 embryos per plate, on 560Y medium for 4
hours and then aligned within the 2.5-cm target zone in preparation for
bombardment.

[0313] Each reagent is added sequentially to the tungsten particle
suspension, while maintained on the multitube vortexer. The final mixture
is sonicated briefly and allowed to incubate under constant vortexing for
10 minutes. After the precipitation period, the tubes are centrifuged
briefly, liquid removed, washed with 500 ml 100% ethanol, and centrifuged
for 30 seconds. Again the liquid is removed, and 105 .mu.l 100% ethanol
is added to the final tungsten particle pellet. For particle gun
bombardment, the tungsten/DNA particles are briefly sonicated and 10
.mu.l spotted onto the center of each macrocarrier and allowed to dry
about 2 minutes before bombardment.

[0314] Particle Gun Treatment

[0315] The sample plates are bombarded at level #4 in particle gun #HE34-1
or #HE34-2. All samples receive a single shot at 650 PSI, with a total of
ten aliquots taken from each tube of prepared particles/DNA.

[0316] Subsequent Treatment

[0317] Following bombardment, the embryos are kept on 560Y medium for 2
days, then transferred to 560R selection medium containing 3 mg/liter
Bialophos, and subcultured every 2 weeks. After approximately 10 weeks of
selection, selection-resistant callus clones are transferred to 288J
medium to initiate plant regeneration. Following somatic embryo
maturation (2-4 weeks), well-developed somatic embryos are transferred to
medium for germination and transferred to the lighted culture room.
Approximately 7-10 days later, developing plantlets are transferred to
272V hormone-free medium in tubes for 7-10 days until plantlets are well
established. Plants are then transferred to inserts in flats (equivalent
to 2.5" pot) containing potting soil and grown for 1 week in a growth
chamber, subsequently grown an additional 1-2 weeks in the greenhouse,
then transferred to classic 600 pots (1.6 gallon) and grown to maturity.
Plants are monitored and scored for and altered level of expression of
the WRKY sequence of the invention. Alternatively, the WRKY activity can
be assayed (i.e., enhance disease resistance).

[0321] For Agrobacterium-mediated transformation of maize with a WRKY
polynucleotide operably linked to ubiquitin promoter, preferably the
method of Zhao is employed (U.S. Pat. No. 5,981,840, and PCT patent
publication WO98/32326; the contents of which are hereby incorporated by
reference). Briefly, immature embryos are isolated from maize and the
embryos contacted with a suspension of Agrobacterium, where the bacteria
are capable of transferring the WRKY nucleotide sequences to at least one
cell of at least one of the immature embryos (step 1: the infection
step). In this step the immature embryos are preferably immersed in an
Agrobacterium suspension for the initiation of inoculation. The embryos
are co-cultured for a time with the Agrobacterium (step 2: the
co-cultivation step). Preferably the immature embryos are cultured on
solid medium following the infection step. Following this co-cultivation
period an optional "resting" step is contemplated. In this resting step,
the embryos are incubated in the presence of at least one antibiotic
known to inhibit the growth of Agrobacterium without the addition of a
selective agent for plant transformants (step 3: resting step).
Preferably the immature embryos are cultured on solid medium with
antibiotic, but without a selecting agent, for elimination of
Agrobacterium and for a resting phase for the infected cells. Next,
inoculated embryos are cultured on medium containing a selective agent
and growing transformed callus is recovered (step 4: the selection step).
Preferably, the immature embryos are cultured on solid medium with a
selective agent resulting in the selective growth of transformed cells.
The callus is then regenerated into plants (step 5: the regeneration
step), and preferably calli grown on selective medium are cultured on
solid medium to regenerate the plants.

EXAMPLE 6

Soybean Embryo Transformation

[0322] Soybean embryos are bombarded with a plasmid containing a WRKY
polynucleotide operably linked to a Scp1 promoter (U.S. Pat. No.
6,072,050) as follows. To induce somatic embryos, cotyledons, 3-5 mm in
length dissected from surface-sterilized, immature seeds of the soybean
cultivar A2872, are cultured in the light or dark at 26.degree. C. on an
appropriate agar medium for six to ten weeks. Somatic embryos producing
secondary embryos are then excised and placed into a suitable liquid
medium. After repeated selection for clusters of somatic embryos that
multiplied as early, globular-staged embryos, the suspensions are
maintained as described below.

[0323] Soybean embryogenic suspension cultures can be maintained in 35 ml
liquid media on a rotary shaker, 150 rpm, at 26.degree. C. with
florescent lights on a 16:8 hour day/night schedule. Cultures are
subcultured every two weeks by inoculating approximately 35 mg of tissue
into 35 ml of liquid medium.

[0324] Soybean embryogenic suspension cultures may then be transformed by
the method of particle gun bombardment (Klein et al. (1987) Nature
(London) 327:70-73, U.S. Pat. No. 4,945,050). A Du Pont Biolistic
PDS1000/HE instrument (helium retrofit) can be used for these
transformations.

[0325] A selectable marker gene that can be used to facilitate soybean
transformation is a transgene composed of the 35S promoter from
Cauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), the
hygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;
Gritz et al. (1983) Gene 25:179-188), and the 3' region of the nopaline
synthase gene from the T-DNA of the Ti plasmid of Agrobacterium
tumefaciens. The expression cassette comprising the WRKY sequence
operably linked to the Scpl promoter can be isolated as a restriction
fragment. This fragment can then be inserted into a unique restriction
site of the vector carrying the marker gene.

[0326] To 50 .mu.l of a 60 mg/ml 1 .mu.m gold particle suspension is added
(in order): 5 .mu.l DNA (1 .mu.g/.mu.l), 20 .mu.l spermidine (0.1 M), and
50 .mu.l CaCl.sub.2 (2.5 M). The particle preparation is then agitated
for three minutes, spun in a microfuge for 10 seconds and the supernatant
removed. The DNA-coated particles are then washed once in 400 .mu.l 70%
ethanol and resuspended in 40 .mu.l of anhydrous ethanol. The
DNA/particle suspension can be sonicated three times for one second each.
Five microliters of the DNA-coated gold particles are then loaded on each
macro carrier disk.

[0327] Approximately 300-400 mg of a two-week-old suspension culture is
placed in an empty 60.times.15 mm petri dish and the residual liquid
removed from the tissue with a pipette. For each transformation
experiment, approximately 5-10 plates of tissue are normally bombarded.
Membrane rupture pressure is set at 1100 psi, and the chamber is
evacuated to a vacuum of 28 inches mercury. The tissue is placed
approximately 3.5 inches away from the retaining screen and bombarded
three times. Following bombardment, the tissue can be divided in half and
placed back into liquid and cultured as described above.

[0328] Five to seven days post bombardment, the liquid media may be
exchanged with fresh media, and eleven to twelve days post-bombardment
with fresh media containing 50 mg/ml hygromycin. This selective media can
be refreshed weekly. Seven to eight weeks post-bombardment, green,
transformed tissue may be observed growing from untransformed, necrotic
embryogenic clusters. Isolated green tissue is removed and inoculated
into individual flasks to generate new, clonally propagated, transformed
embryogenic suspension cultures. Each new line may be treated as an
independent transformation event. These suspensions can then be
subcultured and maintained as clusters of immature embryos or regenerated
into whole plants by maturation and germination of individual somatic
embryos.

[0329] The above examples are provided to illustrate the invention but not
to limit its scope. Other variants of the invention will be readily
apparent to one of ordinary skill in the art and are encompassed by the
appended claims. All publications, patents, and patent applications cited
herein are indicative of the level of those skilled in the art to which
this invention pertains. All publications, patents, and patent
applications are hereby incorporated by reference to the same extent as
if each individual publication or patent application was specifically and
individually indicated to be incorporated by reference.